NBSIR 86-3345

NAT'L INST. OF STAND & TECH

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1986 MEETING OF THE

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ELECTROPHORESIS SOCIETY

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MARCH 26-28, 1986

PROCEEDINGS

Editor: Dennis J. Reeder

SPONSORED DY:

U.S. DEPARTMENT OF COMMERCE

National Bureau of Standards

National Measurement Laboratory NATIONAL BUREAU OF STANDARDS

Center for Analytical Chemistry _

Gaithersburg, MD 20899 AND

THE ELECTROPHORESIS SOCIETY

March 1986

QC

100

.U56

NO. 86-3345

1986 a. 3-

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary

National Bureau of Standards, Ernest Ambler, Director

NBSIR 86-3345

i#-: A 986 MEETING OF THE

/ AMERICAS BRANCH

. OF THE

ELECTROPHORESIS"SOCIETY

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MARCH 26-28, 1986

PROCEEDINGS

Editor: Dennis J. Reeder

SPONSORED DY:

U.8. DEPARTMENT OP COMMERCE

National Bureau of Standards

National Measurement Laboratory NATIONAL BUREAU OF STANDARDS

Center for Analytical Chemistry _

Galthersbu rg. M D 20899 A

N

U

THE ELECTROPHORESIS SOCIETY

March 1986

U.S. DEPARTMENT OF COMMERCE, Malcolm Baldrige, Secretary

National Bureau of Standards, Ernest Ambler, Director

INTRODUCTION

The Electrophoresis Society (America's Branch) in 1985 had the task of deciding

whether to hold a national meeting in 1986, the same year as the planned

international meeting in London. As the council met, the consensus was that

there would be sufficient interest in a meeting that plans should be made. The

authors of the plenary sessions were contacted and graciously committed to the

task of writing papers for publication in the proceedings.

The meeting format was agreed upon to allow maximum interactions among the

participants, to allow sufficient time for plenary lecturers to give a comprehensive lecture, and to enable the attendees to have a published document

for later reference.

The afternoon meetings were structured to permit the greatest information

exchange by arranging sessions on selected topics, moderated by session

chairpersons who would facilitate the direction of the presentations. The

cooperation and willingness of individuals to participate would be the key to a successful information exchange. The informal nature of the sessions was devised to allow the expert and the novice to exchange findings and details

about their research.

The impact of electrophoretic separations on biochemical analyses is reflected

in the topics covered in this publication. Two-dimensional electrophoresis (2-

DE) has matured to become an accepted tool for clinical studies. Immobilized

pH gradients are becoming more reproducible and have found use as the first

dimension high resolution separation step in 2-DE. The chemistry of protein

detection in gels has progressed significantly to enable quantification.

Although not fully understood, studies of staining mechanisms are proceeding.

Nucleic acid separations in gels is a common technique, but the theoretical

aspects of the separation process are just now being explored. Standardization

in electrophoretic separations is addressed in relation to new materials for

molecular weight, size and charge standards. The beginnings of interlaboratory

standardization by round-robin studies also is reported. New matrix materials

for separations are introduced . Finally, the practical applications of many

techniques are reported.

II
'
ACKNOWLEDGEMENTS

This publication was compiled from papers and abstracts submitted

for the Electrophoresis Society Meeting. I am particularly

grateful to Jesse J. Edwards and Margaret C. Kline for their work

in the final days of preparation. For helpful discussions, I am

also indebted to Diane K. Hancock, Kathy C. Stang and Kathleen D. Kilmer

.

DISCLAIMER

The use of trade names and manufacturer's names in this publication

in no way represents an endorsement of a particular product or manufacturer by the National Bureau of Standards or the

Electrophoresis Society.

iii
CONTENTS

PLENARY PAPERS:

Bruce Budowle

Making Ultrathin- layer Polyacrylamide Gel Isoelectric Focusing a Reproducible Methodology

John Fawcett 13 Polyacrylamide Gel Electrofocusing in pH gradients formed by Carrier

Ampholytes and by Immobiline: Pro's and Con's

Carl R. Merril 36 Stains for the Detection of Proteins Separated by Electrophoretic

Methods

Susan C. Olson and Carl R. Merril 66 Peptide Separations: Techniques Reviewed

Philip Serwer 86 Viruses and Viral Components in Extracts of Infected Cells:

Analysis by Agarose Gel Electrophoresis

Nancy Stellwagen 10* Gel Electrophoresis of DNA

* N. L. Anderson's paper on Macromolecular Identification on Two-Dimensional Gels was not received in time for publ icat ion

.

ABSTRACTS:

Stephen H. Blose 1J7 The Functions of a Protein Database Developed from Comput e r - Ana 1 v/.ed

Quantitative Two-Dimensional Gels

Yvon C . Chagnon

Genetic Study of Marine Vertebrate and Invebrate Populations Using

Ultra-Thin Isoelectric Focusing Techniques

Gail Chuck and George Hug 1 J9 Detection of Hexosaminidase Isozymes by Slab Gel Isoelectric Focusing

(PAG-IEF)

Richard B. Cook

NuFix Glyoxyl Agarose: A Versatile New Medium for Electrophoresis,

Blotting and Chromatography

Dale Dykes

The Use of Biotinylated Human DNA Probes for Detect inr. P. FhP'.s

Anne M. Gambel, Bruce Budowle, and Randall S. Murch

Alpha 1-Antitrypsin (PI) Phenotyping by Ultrathin haver I socle, trio

Focusing
James I. Garrels and Robert Franza, Jr 143

New Development in Hardware and Software for 2D Gel Analysis .

Douglas M. Gersten, E.J. Zapolski, T. J. Golab, and R.S. Ledley . 144

Automated Nucleic Acid Electrophoresis and Hybridization Analysis

Robert L. Gilman • • 1^5

Electrophoretic Separation of High Density Lipoprotein Fractions

E. Gombocz , D. Tietz, S. Hurtt, and A. Chrambach 146

Physical Characterization of Turnip Crinkle and Pellargonium

Flower Break Viruses by Agarose Gel Electrophoresis:

Applicability of Polystyrene Size Standards and of

Moving Boundary Electrophoresis Buffer Systems

Angelika Gorg, W. Postel, S. Gunther, and J. Weser . 147

Immobilized pH Gradients for One- and Two-Dimensional Separations.

Applications

Samir M. Hanash, L.J. Baier, and Rork Kuick 148

A New Prognostic Indicator in Acute Lymphoblastic Leukemia Detected

by Two-Dimensional Polyacrylamide Gel

Electrophoresis (2-D PAGE)

Diane K. Hancock 149

Mechanistic Studies of Silver Staining

William E. Heydorn, G. J. Creed, C. R. Creveling and

D.M. Jacobowitz 150

Studies on Catechol-O-Methyltransferase in Rat Brain using TwoDimensional Gel Electrophoresis

Ann E. Kaplan and G.N. Gray 151

Immobiline Electrophoresis: pi Values for Proteins of LDH-4 and -5

from Control and Nitrosomethylurea-Transformed

Neoplastic Cell Lines

Reinhold C. Mann, B.K. Mansfield, and J. K. Selkirk 152

Parametric Versus Non-Parametric Gel Image Analysis

Betty K. Mansfield, R. C. Mann, and J. K. Selkirk ...... 153

2 , 5 -Diphenyloxazole (PPO) in Dimethyl Sulfoxide (DMSO) Regeneration

for Re-Use in Fluorography

Mark Miller and S.S. Thorgeirsson 154

Analysis of Micro -Variations in the Rate of Synthesis of Individual

Polypeptides Isolated from Cultures of

the Rat Hepatoma Cell Line H4-II-E as Measured by

Quantitative Two-Dimensional Gel Electrophoresis

V
Rodger Morrison and M.G. Brown 155 Beta-Hemoglobin Polymorphism in White-Tailed Deer:

Suitability and Application to Wildlife Law Enforcement

James E. Myrick, M.K. Robinson, I.L. Hubert, C.J. Bell,

and S. P. Caudill

Preparation of Urine and Serum Samples with Centrifugal

Microconcentrators and Total Protein Analysis by

Ultraviolet-Difference Spectroscopy for Two-Dimensional

Electrophoretic Analysis

Stephen Peats, S. Nochumson, F.H. Kirkpatrick

Effects of Borate on Agarose Gel Structure

Kristy L. Richie and D.J. Reeder

Results of an Interlaboratory Round Robin Using Isoelectric Focusing

to Assess Reproducibility of Rehydratable Gels

Atul Sahai and S. M. Hanash

Elevated Levels of p21 ras Detected in Blast Cells from Leukemic

Patients

David Sammons and R. Humphreys

Intermediate Range Molecular Weight Markers for SDS Gels

Burton E. Sarnoff, D. Berretta and P. Todd

The Effect of Calcium Ion Concentration on the Electrophoretic

Mobility of Two Cultured Human

Embryonic Kidney Cell Populations

Cheri Seitz

Advantages of Focusing Carbamylation Trains in a Flat -Bed System

John R. Strahler, S. Hanash, L. Somerlott and A. Gorg

Two-Dimensional Polyacrylamide Gel Electrophoresis of Lymphocyte

Proteins with Immobilized pH Gradients

in the First Dimension

Dietmar Tietz, E. Gombocz and A. Chrambach

Physical Characterization of Particles and Gel Fibers on the Basis of

Non-Linear Ferguson Plots in Agarose Gel

Electrophoresis

Kaija H. Valkonen and D. Goldman

Preparative and Analytical Immobiline Electrofocus i ng of 1 lass 111

Hepatic Alcohol Dehydrogenase
Peter J. Wirth and S. S. Thorgeirsson 166

Sequential Analysis of Chemically Induced Hepatoma Development Using

Two-Dimensional Electrophoresis

(2-D PAGE)

Virginia P. Wray 167

Foundation Experiments for Building a Protein Database

Joseph Yudelson 168

Mechanism of Nickel and Silver Visualization of

Protein Electrophoregrams

Rashid A. Zeineh, J. Zeineh and M. Zeineh 169

Comparison of 2 -Dimensional Gel Electrophoretic Separations

Using Soft Laser Scanning Densitometry with Computer

Color Graphics

Rashid A. Zeineh, J. Zeineh and M. Zeineh 170

Multilane Strip -Chart Printing Program for DNA

Sequence Reading

vii

PLENARY PAPERS

MAKING ULTRATHIN-LAYER POLYACRYLAMIDE GEL ISOELECTRIC

FOCUSING A REPRODUCIBLE METHODOLOGY

Bruce Budowle

Forensic Science Research and Training Center, Laboratory

Division, FBI Academy, Quantico, Virginia 22135

Introduction

Recent advances in isoelectric focusing have shown that

proteins can be separated on ultr athin-layer polyacrylamide gels

(<_ 0.36 mm thick gels) (1). The advantages of ult rathin-layer

polyacrylamide gel isoelectric focusing (ULPAGIF) are increased

resolution and sensitivity of detection, shorter separation and

staining times, and reduced cost. Ultr athin-layer gels have a greater surface area-to-volume ratio than thicker gels and,

therefore, are more effective at heat dissipation. Thus, higher

voltages can be applied to the gels and an increased

concentration of synthetic carrier ampholytes (4%, w/v) can be

incorporated into the gel with the expectation of superior

resolution. Furthermore, the higher voltage gradient produces

narrower protein bands (2, 3), which results in more protein per

unit gel volume. Since this, in effect, presents more protein

for the subsequent assay, the sensitivity of detection of the

system is increased. The applied higher field strengths also

result in faster separation times making it possible to type more

samples in less time than previously possible. Budowle (3-9) has

shown that the separation times by ULPAGIF for the genetic

markers phosphoglucomutase- 1 (PGM), erythrocyte acid phosphatase

1
(EAP), alpha 1 -antitrypsin (Pi), group-specific component (Gc),

transferrin (Tf), esterase D (EsD) and hemoglobin (Hb) can be as fast as 37, 22, 75, 125, 135, 53 and 25 minutes, respectively.

Due to shorter diffusion pathways, assay times [such as for

silver staining (10)] are greatly reduced. Rapid staining is an important consideration particularly for functional assays for

polymorphic enzymes where diffusion of the isozyme patterns can

reduce resolution. Finally, by reducing the gel thickness, the

quantities of reagents (and thus the cost) are reduced without

compromising the number of samples analyzed.

Despite the advantages of using ultra thin-layer gels,

there has been a reluctance to routinely use this methodology for

genetic marker typing. Part of this reluctance can be attributed

to a lack of reproducibility of banding patterns from gel to gel

and lack of linearity across a gel. Problems with variation in

resolution and wavy patterns can lead to possible mistypings,

increased inconclusive determinations and time-consuming retyping of samples. These problems are surmountable provided

certain considerations are incorporated into ULPAGIF protocols.

A) . Casting Techniques

Care should be exercised in the manner the gels are

cast. There are two general methods for casting ultrathin-layer

polyacrylamide gels - capillary and flap techniques [see Allen

(11) and Radola (12) for the methodology]. After gels are poured

using the flap technique, a weight is placed on the top glass

plate to seal the gel. If this weight is not evenly distributed,

the resulting gel will not have a uniform thickness. The results

o
are distorted protein patterns and undesired gel drying.

Therefore, several glass plates should be used to evenly

distribute the weight (4).

The capillary method in comparison to the flap

technique is less time-consuming and allows for easier gel

preparation. The major problem with the capillary method is

avoiding the production of bubbles in the gel during pouring.

This problem can be solved by lightly tapping the plate at the

gel solution front as it is migrating under the plate by

capillary action. This will produce an even flow of the gel

solution and eliminate bubble formation. In the event that a bubble is trapped under the glass plate, simply slide the glass

plate to expose the bubble, the bubble will dissipate, and then

slide the plate back to its original position. After the gel

polymerizes remove it from the casting tray and wrap it in

plastic wrap. Gels can be maintained in the refrigerator in this

condition from one to three weeks. In contrast, gels cast by the

flap technique can be maintained at room temperature from three

to six months (unpublished data).

The methods developed by Budowle (3-9) routinely used

200 urn thick gels instead of thinner gels used by others (12-18).

Even with the best of care in casting gels, there are still some

slight variations in thickness within a gel. A 10% decrease in a portion of a 200 urn thick gel (20 urn) is less deleterious than a

10% decrease in a portion of a 100 urn thick gel (10 urn). The

latter gel, at times, yielded uneven migration of bands, resulted

3
in protein migrating along the gel surface instead of entering

the gel and/or dried out (unpublished data and personal

communication, R. C. Allen).

B

)

. Effect of Electrode Distance

As the distance between the electrodes is reduced the

minor depressions in the gel present less of a problem (due to

increased conductivity). Kinzkofer and Radola ( 19 ) have had

great success with 50-100 urn thick gels with inter-electrode wick

distances of 1-3 cm. However, for the purposes of genetic marker

typing these inter-electrode wick distances are impractical. The

pH ranges of commercially available synthetic carrier ampholytes

are not narrow enough for such distances and thus impose

limitations on gel dimensions. Allelic products with slightly

different isoelectric points lie too close together to resolve in

this manner, especially when functional enzyme overlay assays are

employed (diffusion problems). When the in ter-elec trode wick

distance is 5-10 cm, as is necessary for most genetic marking

typing systems, 200 urn thick or thicker gels should be employed.

Use of a gel with this thickness will contribute favorably to the

reproducibility of the system.

C) . Effects of Sample Protein and Salt Concentrations

Excess protein loading is tolerated more by thicker

gels than thinner ones. For example, in PGM analysis, 6-8 ug of

protein can be applied to 200 urn thick gels with an 8 cm interelectrode wick distance containing 4% (w/v) pH 5-7 ampholytes

(LKB) and produce linear PGM subtype patterns (20, 21). Less

than half that protein concentration can be applied to a similar

4
gel, 100 um thick, without producing bowed PGM subtype patterns.

Similar trends occur for salt loading. However, in the case of

salts, diluting the sample (and still applying the whole sample

to the gel) can reduce deleterious salt effects. It appears the

limiting factor is the amount of salt per unit sample volume and

not the total amount of salt. The effect of salts led Pflug (21)

to develop a wedge-shaped gel for PGM subtyping of semen samples

.

The gels were 300 um thick at the anode decreasing in thickness

to 50 um at the cathode. Pflug applied semen stain extracts at

the thicker portion of the gel. There was less distortion of PGM

due to the salt effects. The wedge-shaped gel combined the

higher resolving capacity of ultr athin-layer gels with the

loading capacity of thicker gels. In contrast, Budowle , et al

.

(22), utilizing a straight 200 um thick gel, a reduced interelectrode wick distance of 8 cm and a much higher final voltage

gradient (290 V/cm vs. 180 V/cm) , observed no deleterious salt

effects on PGM from semen stains.

It should be noted that protein loading tolerances are

also dependent upon the pH range and the particular manufacturer

of the synthetic carrier ampholytes. For example, more than

twice the protein that can be applied to a pH 5-7 ampholyte (LKB)

gel can be applied to a pH 4. 5-5. 5 Servalyte gel without any

protein band distortion. While a gel containing pH 4. 5-5.

4

Pharmalytes can only handle 70% of the protein of the Servalyte

gel (unpublished data).

5
D) . Effect of Field Strength

The primary advantage of ultrathin-layer polyacrylamide

gels is the ability to use higher field strengths. Rilbe (23),

Giddings and Dahlgren (24), and Allen (11) have shown that

resolution is proportional to the square root of the voltage

gradient. Since these gels can more effectively dissipate Joule

heat, higher field strengths can be utilized with the expectation

of increased resolution. For gels with an inter-electrode

distance of 5-10 cm, voltage gradients as high as 300 - 700 V/cm

have been used (2-12, 20, 22). Furthermore, the higher voltage

gradient can produce narrower protein bands (2, 3). The

concentrating of protein bands into more narrow zones results in

more protein-per-uni t gel volume which, in effect, presents more

protein for the subsequent assay. Allen and Arnaud (2) using the

Rohament P enzyme test demonstrated that a ten-fold voltage

gradient increase (50- 500 V/cm) yielded a 2.3-fold increase in

the number of bands resolved and a band-width decrease by a factor of 2.3 to 2.5. Budowle and Murch (3) observed that by

increasing the voltage gradient from 340 V/cm to 460 V/cm a onethird decrease in the width of Pi bands could be realized. Many

investigators (14-18) have utilized ULPAGIF, but do not take

obvious advantage of the application of higher field strengths.

Usually these investigators used voltage gradients less than 200

V/cm. Genetic variants with very close isoelectric points, such

as the Cl and C3 allelic products of Tf (25-27), the M3 and M2

6
allelic products of Pi (28), and rare variants of Hb , can be

easily resolved when higher field strengths are employed

(300 V/cm - 700 V/cm)

.

A problem that manifests itself with the use of certain

ampholyte ranges and high field strengths is the appearance of

"hot spots" (29). These "hot spots" are conductivity gaps in the

gradient and serve as a limiting factor for the field strength

that may be applied to the gel. With sufficiently high field

strengths the "hot spots" become burn spots on the gel (9, 11).

This is an undesirable effect, especially when the protein(s) of

interest has yet to be resolved. The problem can be solved by

adjusting one of the electrodes (after a prescribed time into the

run) to bypass the area of the gel where the conductivity gap

exists. There then will be a shorter inter-electrode wick

distance, a more uniform conductance across the gel, no gel

burning, and the run can be completed to obtain the desired

resolution. This approach has been successfully used for

subtyping Gc in bloodstains (9).

E )

.

Reproducing the Voltage Gradient

Not only is resolution dependent upon the voltage

gradient, but for ul tr athin-layer polyacrylamide gels, the

voltage gradient is also important for gel-to-gel reproducibility

and linearity across a gel. Budowle (7, 20) demonstrated that

once the ideal voltage gradient conditions were empirically

determined, the voltage gradient had to be reproduced at

intervals for every ULPAGIF run. Although the gels were still
focused with constant power, the power mode was adjusted at

regular intervals depending upon the voltage parameter. By

following this approach, the same results were obtained from gel

to gel. To produce band linearity across a gel, it was found

that the initial voltage (regardless of the inter-electrode wick

distance) applied to the gel during prefocusing could not exceed

250 volts. As long as the conditions were reproduced, wellresolved, highly reproducible patterns were obtained. Further,

this approach appears to compensate for ambient temperature and

humidity effects. Current can also be a limiting factor.

However, the previously reported methods (7, 20) have maintained

the current at low enough levels so as not to have an impact on

gel reproducibility.

F ) . Reagents

Fresh stock solutions of recrystallized acrylamide

should be considered for ULPAGIF. Chrambach, et al. (30) have

shown that impurities, such as acrylic acid, will confer ion

exchange properties to the gel. This can cause irr eproduc ibi 1 i ty

as well as artifacts between runs. Budowle (6) observed that

lower grades of acrylamide appear to inhibit EsD activity. This

is presumably analogous to the effects of impure acrylamide on

mouse plasma esterases reported by Allen, et al. (31). Fresh

stock solutions of acrylamide (less than one month old) should be

used to avoid distortions originating in the anodal portion of

the ultr athin-layer polyacrylamide gel. Older stock solutions

cause distortion in the gel resulting in waviness across the gel

8
so that similar proteins in different sample lanes can not be

compared effectively. In addition, when a functional assay is

utilized to visualize the polymorphic enzymes, the gels should

sit overnight prior to use. Freshly poured gels tend to inhibit

enzymatic activity. This phenomenon was readily observed for EsD

and in particular for EAP (5, 6). The activity of the C band of

EAP was completely inhibited in polyacrylamide gels that

polymerized for only one to three hours. Thus, using fresh

recrystallized acrylamide stock solution and permitting

polyacrylamide gels to sit overnight prior to use are imperative

for minimizing mistypings of genetic marker systems.

If the waviness which originates at the anode still

persists, it is suggested to employ alternative anolytes. The

anolyte for pH 5-7 ampholyte (LKB) gels for successful subtyping

of PGM is phosphoric acid. However, when phosphoric acid is used

as the anolyte with narrow-range Pharmalytes, an anodal

distortion occurs. By using saturated L-aspartic acid instead of

phosphoric acid the distortion is greatly reduced (9). Also,

some narrow-range ampholytes which yield good separation of

genetic variants, (such as Pharmalyte pH 4. 2-4. 9 for Pi (3),

pH 4. 5-5. 4 for Gc (9, 32), and pH 6. 7-7. 7 for Hb (8)) may still

exhibit an inherent instability in the gradient. To alleviate

this waviness enhancing these narrow range ampholytes with small

amounts (1:10) of wider range ampholytes (pH 4-6, pH 4-6.5 or

pH 3-10) is required (8, 9). In addition to yielding more linear

9
patterns across a gel, enhancing the pH gradient with wider pH

range ampholytes does not appear to compromise the separation

distances between allelic variant bands.

There is one constraint on this methodology which is

out of the control of the laboratory. This is the batch-to-batch

variation of ampholytes. To subtype Tf (7) pH 5-7 ampholytes

(LKB) are used. While lots 48 and 50 produced the desired

patterns demonstrated in the literature, it was impossible to

resolve the C1C3 phenotype when lot 49 was used (unpublished

data). These problems have been observed for Servalyte and

Pharmalyte as well. To avoid these problems in the future

ampholyte manufacturers will have to take responsibility for

quality control.

Following the approaches described above, reliable,

reproducible, and linear band patterns can be achieved using

ULPAGIF for genetic marker typing as well as for most isoelectric

focusing protein analyses. This is of particular concern for

laboratories with heavy case loads. Time and expenses taken to

rerun samples may be prohibitive. Further, when sample

size is a limiting factor, as can be the case for forensic

samples or supplies of extremely rare variants, multiple tests to

obtain an analysis may prove to be impossible. If care is taken,

seldom will a gel have to be rerun.
References

1) Gorg, A., Postel, W. and Westermeier, R., Anal. Biochem.

1978, 89, 60-70.

2) Allen, R. C. and Arnaud, P., Electrophoresis 1983, 4,

205-211

.

3) Budowle, B. and Murch, R. S. , Electrophoresis, 1985, 6,

523-525.

4) Budowle, B. , Biotechniques 1985, 3, 92-93.

5) Budowle, B., Electrophoresis 1984, 5, 254-255.

6) Budowle, B. , Elec trophoresis 1984, 5, 314-316.

7) Budowle, B., Electrophoresis 1985, 6, 97-99.

8) Budowle, B. and Eberhardt, P. , Hemoglobin (in press).

9) Budowle, B., Electrophoresis (in press).

10) Budowle, B. and Scott, E., For. Sci. Int . 1985, 28, 269-275

11) Allen, R. C., Electrophoresis 1980, 1, 32-37.

12) Radola, B. J., Electrophoresis 1980, 1, 43-56.

13) Radola, B. J., in Radola, B. J. (Ed.), Electrophoresis 1980

Walter de Gruyter , Berlin 1980, pp. 79—93

14) Goedde, H. W„, Benkmann, H. G. and Hirth, L., Hum. Genet.

1981, 57, 434-436.

15) Pflug, W. , Vigne, U. and Bruder , W., Electrophoresis 1981,

2, 327-330.

16) Divall, G. B. and Ismail, M. , For. Sci. Int. 1983, 22,

253-263.

17) Gill, P. and Sutton , J . G .

,

Elec trophores i

s

1985, 6, 23-26

18) Gill, P. and Sutton , J . G .

,

I U / U 1 J. i, ji o QUU J U U W U ii

y U o VJ • y Electrophoresis LlC'vvl U^Uvl C O 1 O 1985, I y U J | 6,W y 274-279.

19) Kinzkofer, A. and Radola, B. J., Electrophoresis 1981,

2, 174-183.

20) Budowle, B., Elec trophoresis 1984, 5, 165-167.

21) Pflug, W., Electrophoresis 1985, 6, 19-22.
22) Budowle, B. , Murch, R. S., Davidson, L. C., Gambel, A. M.

and Kearney, J. J., J. For. Sci. (in press).

23) Rilbe , - H.. Ann . N.Y. Acad. Sci. 1973, 209, 1 1-22.

24) Giddings, J. C. and Dahlgren, H., Sep. Sci. 1971, 6, 345-356

25) Kuhnl, P. and Spielmann, W. , Hum. Genet. 1978, 43, 91-95.

26) Kuhnl, P. and Spielmann, W., Hum. Genet. 1979, 50, 193-198.

27) Gorg, A., Weser, J., Westermeier, R., Postel, W.

,

Weidinger, S., Patutschnick , W. and Cleve, H., Hum. Genet.

1983, 64, 222-226.

28) Gorg, A., Postel, W. , Weser, J., Weidinger, S.,

Patutschnick, W. and Cleve, H., Electrophoresis 1983, 4,

153-157.

29) Righetti, P. G. and Drysdale, J. W. , Isoelectric Focusing,

Nor th-Holland , Amsterdam 1976, p. 463*

30) Chrambach, A., Jovin, T. M., Svendsen, P. J. and Rodbard, D.

in: Castimpooias , N. (Ed.), Methods of Protein Separation,

Plenum Press, New York 1976, Vol 2., pp. 27-144.

31) Allen, R. C.,"Propp, R. A. and Moore, D. J., Histochem.

Cytochem. 1965, 13, 249-254.

32) Lizana, J. and Olsson, I., J. Clin. Chem. Biochem. 1984,

22, 545-549.

’‘This is publication number 86-3 of the Laboratory Division of

the Federal Bureau of Investigation. Names of commercial

manufacturers are provided for identification only and inclusion

does not imply endorsement by the Federal Bureau of

Investigation . ”
Polyacrylamide Gel Electrofocusing in pH gradients

formed by Carrier Ampholytes and by Immobiline:

Pro's and Con’s.

John S. Fawcett

Andreas Chrambach

Section on Macromolecular Analysis,

Laboratory of Theoretical and Physical Biology,

National Institute of Child Health and Human Development,

National Institutes of Health, Bethesda, MD 20892

Ever since the introduction of immobilized pH gradients (IPG) by

Gasparic et al. (1), one has to make a decision in the laboratory

practice of "charge fractionation" whether to apply

electrofocusing in IPGs (IPGEF) or whether to continue the

isoelectric focusing technique in a pH gradient naturally formed

by the carrier ampholytes (CA) , designated here as IEF. The

answer to that question will depend on the particular application

and relevance of some of the following criteria: 1) Resolving

power; 2) speed; 3) reproducibility; 4) volume, ionic strength

and entry of the sample; 5) experimental simplicity; 6) ease of

selection of the range and shape of the pH gradient; 7)

susceptibility to pH and voltage measurement; 8) perturbation by

gel hydration and dehydration phenomena; 9) artefacts; 10)

compatibility with SDS-PAGE for 2D-separat ions ; 11) ease of

protein isolation, preparative load capacity and purity of
product; 12) equipment needs and cost.

Application of these criteria to IEF and to IPG

electrofocusing (IPGEF) at this time must rest, unfortunately, on

an extremely limited data base as far as IPGEF is concerned,

compared with the many thousands of papers on IEF in the

literature. This imbalance necessarily decreases the reliability

of our conclusions.

Finally, the philosophical point needs to be made that IPGEF,

just because it is the newer method, must not therefore

necessarily supersede IEF. We take it for an axiom that every

method has its proper and optimal area of usefulness, and that no single method is applicable or useful for all separation

problems. The real question is to find these areas of specific

applicability, both for IEF and for IPGEF.

1) Resolving power:

The resolving power in electrofocusing is proportional to the

degree of flatness of the pH gradient as well as to field

strength [equ.138 of (2)]. Narrow pH ranges and therefore

flat pH gradients are applicable as a device promoting resolving

power in those analytical or preparative separation problems

which comprise components with closely distributed pis. When the

mixture to be resolved comprises a wide spectrum of pis, a steep

pH gradient, or a flat pH gradient of great length is required.

The latter is impractical because electrofocusing at high field

strength over a long gel requires impracticably high voltages and

unduly extended focusing time. An attempt to resolve this

dilemma by carrying out electrofocusing at a 45° angle to the

orientation of the pH gradient has failed so far (3)

.

In separation problems to which narrow pH ranges and flat pH

gradients are applicable, both IEF and IPGEF seem capable of

providing pH gradients of approximately 0.5 pH units per 10-20 cm gel length, although the methods used to achieve flat pH

14
gradients are different.

a) In IEF, where only limited narrow pi -range CAs are commercially available, one can create the desired flat pH

gradients in at least one of three ways : i) By preparation,

using electrofocusing or other charge fractionation methods, of a narrower from an available wider pi-range; ii) by introduction of

"separators" with pis in the region of the pH gradient which one desires to flatten (4); iii) by "constituent displacement" (5,6)

defined as the electrophoretic removal of undesirable CAs by

electrofocusing between large catholyte and anolyte reservoirs at

the desired terminal pHs of the gradient. It should be noted,

however, that pH gradient flattening by the latter method is slow

so that the desired pH gradient may not be realized prior to the

deterioration of the gradient through the (usually) cathodic

drift

.

b) IPGEF allows one to compute pH gradients of any degree of

shallowness (7). In a recent example, an IPGEF gradient of 0.01

pH unit/cm was achieved (8). It should be kept in mind, however,

that the computations in view of non-ideal conditions may not be

realistically descriptive of the pH gradient obtained

experimentally in a gel, although they should approximate it.

With a few trial-and-error modifications, one should be able to

set up pH gradients of any degree of flatness in IPGEF. The work

involved in these modifications certainly appears less than that

which is needed for pH gradient flattening in IEF by any of the 3

methods discussed above. In addition to the design of overall

flat pH gradients, IPGEF provides a computational procedure for

allowing the formation of flat regions within a steeper overall

pH gradient (9). Since the method allows for full freedom in the

choice and location of a flat region of the pH gradient, it

presents a considerable advance over local pH gradient flattening

by separators (see above) and the associated trial-and-error

procedures in IEF. In summary, IPGEF appears as the superior way

to achieve high resolving power by pH gradient flattening.
To obtain maximal resolution both in IEF and in IPGEF, voltage

should be raised so as to achieve maximal field strength within

the limits of Joule heat dissipation by the apparatus. The

overall conductance across the pH gradient is less by a factor of

at least 20 in IPGEF as compared to IEF and correspondingly

higher voltages are applied to IPGEF. Applied voltages of up to

1000 V/cm have been reported for IPGEF (10), while only

exceptionally well cooled and thin gels in IEF permit application

of as much as 600 V/cm (11). Values of 20-40 V/cm appear

representative for most applications of IEF. It should be kept

in mind, however, that in view of the unevenness of the

conductance profile, documented at least for the pH-range 4-10 in

IPGEF (12) and in IEF (13), it is the field strength at the point

at which the separated zones are located which defines

resolution, not the overall voltage.

It must also be realized that it is the actual and not the

computed field strength which governs resolution. At least in

the wide pH-range of IPGEF, the predicted (7) and experimental

(12) field strengths vary considerably. In the pH 4-10 IPGEF

system, this variation may be 25-fold within a single pH gradient

(12). Similar variations in field strength obtain in IEF

(13,14). When CAs are introduced into IPGEF, the overall

conductance across the pH gradient increases. Again, this

increase may or may not affect the question whether the local

field strength is higher in ICAPG-EF than in IEF, and thus

whether resolution is higher.

In conclusion, the higher field strength predicted for IPGEF

should provide higher resolution than obtainable in IEF. Whether

this increase in resolution actually occurs, depends on the real,

experimentally measurable, field strength. Increase of

resolution by means of pH gradient flattening is available to

both IEF and IPGEF but more extreme flattening is possible, and

flattening is achieved more easily, in IPGEF than in IEF.
2) Speed:

To compare the speed of IPGEF and of IEF, it is necessary to

determine the time required for identical proteins to reach the

steady-state focused position in a similar pH gradient. Such

determinations were made in our laboratory, where the same mixture of colored proteins was applied to pH 4-10 gradients

formed in IEF, in IPGEF and in IPGEF in presence of CAs

2

(ICAPG-EF) (12). In each case, Vh/L , where L = gel length,

were approximately equal so that the comparison of time was based

on equivalent voltages and gel length (15). The results show

that the relative speeds of the 3 methods vary within not more

than a factor of 2. Within that range, IEF is the fastest

method, followed by ICAPG-EF. IPGEF is the slowest of the 3

methods of electrofocus ing . This is due to the fact that in

spite of the high voltage the low conductance regions in the IPG

gel present bottlenecks to the transport of the protein (and

salt) and thereby delay the attainment of the isoelectric

positions

.

In conclusion, the widely held prejudice that IEF is faster

than IPGEF is not substantiated by the data. The advantage is

marginal, and only appears considerable in view of the delayed

migration, due to electric field perturbations, in the horizontal

slab technique of IPGEF.

3) Reproducibility:

Protein patterns in IEF change continuously as a function of time

and voltage, due to the changes in CA distribution [Fig. 2 of

(16)]. It follows that pattern reproducibility is difficult to

obtain since it depends on critical control of experimental

conditions, inclusive of batch identity of CAs. In contrast to

band position, the pH of the focused zone in IEF is highly

reproducible. However, the methods needed to determine the pH of

1 7
the zone in IEF are sufficiently indirect and/or complex to

reduce the degree of precision of pH measurement. In all but one method of pH determination on the steady-state zone, one needs to

derive pH measurement and stained zone position from parallel

gels or tracks which must be assumed to be identical but in

practice never are. In measuring pH by the contact electrode on horizontal slabs, this assumption does not have to be made, since

the identical track previously analyzed for pH can be

subsequently stained; however, this technique depends on prior

knowledge of the time required for attainment of the

steady-state. Another technique is to measure pH indirectly by

"pi markers" in the gel, but this method is highly inaccurate

(see section 7).

IPGEF, by contrast, provides zone positions at the

steady-state which are constant between experiments and

laboratories within the limits of pH gradient reproducibility.

If one defines that reproducibility of pH gradient for a particular set of Immobiline reagents and a particular mode of pH

gradient formation and polymerization of the IPG gel, a high

degree of reproducibility has been claimed (10,17), although not

statistically verified. But reproducibility of pH gradients

necessarily decreases to the degree that batch variability of

Immobilines and their concentrations exists, and to the degree

that different laboratories, or the same laboratories at

different times, use different gradient making and polymerization

techniques. As yet, a data base for quantitating reproducibility

between laboratories and over time periods does not exist.

It even appears rather unlikely that reproducibility of zone positions between laboratories will ever come to pass, for the zone positions in IPGEF depend on a) Immobiline purity,

concentration and stability on storage either as single reagents

or in a mixture; b) accurate dispensing of Immobiline volumes to

form the mixtures by which the gradient gel is made; c) the

technique of gradient formation and the precision of the internal
dimensions of the cassette; d) the degree of incorporation of

Immobilines into the gel matrix. f) If furthermore gels are stored, either intact or in the dehydrated state, their changes

on storage introduce another element of irreproducibility

.

a) The reproducibility of Immobiline reagents with regard to

both purity and concentration is, of course, entirely out of

control of the investigator and within the responsibility of the

manufacturer, as long as the chemical composition of Immobiline

remains a trade secret. Furthermore, it is not known at this

time to what degree IPGEF patterns depend on Immobiline storage

conditions, such as temperature, the presence of oxygen, pH, as well as on the question whether different Immobilines need to be

stored separately or can be stored in mixture. It is also

important to know whether storage requires the presence of

polymerization inhibitors in some (alkaline ?) or all

Immobiline species, and if yes, what the effect of the inhibitors

on the polymerization of the IPG gel is. The single available

study on Immobiline stability (18) does not answer all of these

questions

.

c) Among techniques to make linear gradients, the use of

2-chamber gradient mixers (19) is prevalent. The major

competitive techniques use computer-directed burets (17) or very

simple pumping arrangements (20). One would a priori assume that

both a rather sophisticated method of gradient formation like the

buret method, or an extremely simple method like the pumping

method, are the most reproducible. Of further importance for

gradient reproducibility are the precision of the cassette

dimensions and the mode of filling (one or several entrance

ports, filling from the top vs. from the bottom). At this time,

the evidence is still insufficient to decide which of the various

modes of cassette filling is optimal. Moreover, the filling of

less than 1 mm thick cassettes consisting of one hydrophilic and

one hydrophobic surface gives rise to a wavy surface of the

rising polymerization mixture during either upward or downward
filling of the cassette.

d) Since polymerization depends on the concentrations of

polymerization catalysts and inhibitors, as well as the

temperature of polymerization and on the concentration and purity

of monomers, a comparison between the reproducibilities of the 3

major gradient making techniques would have to use identical

conditions for polymerization inclusive of the control over all

of these factors. Further totally unknown factors affecting the

composition of the Immobiline copolymer are: i) The question

whether all Immobilines incorporate to an equal degree into

polyacrylamide; ii) the question as to the total degree of

incorporation; iii) the question whether each species is

incorporated evenly or asymmetrically along the polymer chain;

iv) the question whether the incorporated Immobiline groups are able to interact. The presently recommended polymerization

procedure for IPGEF gels has not been developed in response to

those questions but is based solely on a study of polymerization

kinetics of individual Immobiline species to give homopolymers

(21). It is assumed on the basis of that study that each

Immobiline species is incorporated 85% by the recommended

procedure of polymerization in the presence of dissolved oxygen

at 50°C using solely persulfate as a free radical donor. A

reproducible Immobiline gel matrix will depend on quantitative

conversion of all monomers, including Immobilines, into the

polymer. Deaeration of the polymerization mixture and

optimization of initiator species and concentrations should help

to approximate such quantitative conversion. The low reaction

rates of at least some of the Immobilines compared to acrylamide

make it even more imperative to convert monomers to polymer

efficiently: Even a low percentage of residual unpolymerized

acrylamide does not guarantee, in view of these differences in

reaction rates, that a particular Immobiline is incorporated with

anything near the same efficiency.

It is concluded that reproducible zone positions and patterns

20
are exclusively obtainable by IPGEF and ICAPG-EF, not by IEF.

However, statistical data concerning gradient and pattern

reproducibility in IPGEF are not available as yet. Certainly,

the advent of commercially prepared Immobiline gels should shift

the burden of reproducibility into the realm of industrial

quality control and be able to at least provide batches of

relatively highly reproducible pH gradient gels.

4) Volume, ionic strength and entry of the sample:

As a first approximation, sample volume limitations appear

apparatus dependent and independent of the selection of either

IEF or IPGEF as a focusing method. In both methods, applied in

either vertical slabs or tubes, there exists no limit to sample

volume in the ideal case. In reality, the instability of the pH

gradient in IEF does set such a limit, however, for 2 reasons:

The sample can migrate into the gel only as long as the changing

pH at the gel terminus provides sufficient mobility at the

appropriate side of its pi. Secondly, the pH profile must remain

steep enough to allow for an approximation of the isoelectric

zone by the sample. By contrast, the stable pH gradient in IPGEF

is not only compatible with large sample volumes, but even with a continuous introduction of sample into the system. This

preparative advantage has not been exploited as yet. The method

may be limited in IPGEF by inadequate migration rates out of the

sample zone (see below) . This problem may be remedied by the

accelerated transport in ICAPG-EF, however possibly at the price

of swelling problems (section 8).

Horizontal slab apparatus is rather limited in regard to

sample volume. Most horizontal apparatus is being used with

sample volumes of 2 to 50 yl depending on gel thickness. This

limitation is largely independent of the technique in sample

application which in IEF is preponderantly by paper strip onto

the gel surface, and in IPGEF by sample slots. To avoid the

21
inconvenience of slot forming during polymerization, a silicone

rubber strip with intermitttent ly spaced holes placed onto the

gel surface has also been found useful in IPGEF (17).

In principle, IPGEF suffers from less restrictions in sample

ionic strength than IEF (10). The rationale for that advantage

is that in IEF the electrophoretic removal of salt leads to a displacement of CAs and consequent shift of the pH gradient.

This mechanism obviously cannot apply to IPGEF where charged

groups are fixed. In reality, however, the limited transport and

the very low current in IPGEF does not allow for an effective

desalting of the sample, with the consequence that protein

migration from the sample zone is retarded. Furthermore, on horizontal slabs, the discrepancy between the conductance of the

sample zone and the surrounding gel is increased at elevated

ionic strength of the sample, so that the distortion of the

electric field due to that discrepancy increases. Since in gel

tubes or in the corresponding isolated tracks of a horizontal

slab (10,22) the field is forced to pass through the sample zone,

the desalting efficiency is increased when IPGEF is carried out

in those modes. The high tolerance for sample ionic strength

expected for IPGEF is only realized in ICAPG where conductance is

sufficient for an effective removal of the salt from the sample

zone, with generation of high enough voltage in the sample zone

to allow for sample entry into the gel.

IPGEF appears to present problems of sample entry. These

problems have led to a practice of loading several times more protein onto otherwise equivalent IPGEF and IEF gels. In part,

these problems probably derive from the zone spreading and the

consequent dilution of the protein within the zone which is due

to the distortion of the electric field passing through a more conductive protein zone located within a less conductive gel. As

already mentioned, this effect can to some degree be reduced by

cutting slab gels into separate tracks, or by use of gel tubes.

A second relevant factor is the inadequate rate of ion transport

22
in a medium of exceedingly low conductance. Furthermore, the low

ionic strength of the medium leads to protein aggregation and

insolubility , an effect that possibly may be remedied by the

addition of CAs or detergents to the sample (23).

Non-quantitative sample entry is not usually encountered in IEF,

and is overcome by the addition of CAs in ICAPG-EF.

5) Experimental ease:

There can be no question that IEF excels in the ease of

experimental procedure over IPGEF, considering that in IEF the pH

gradient forms automatically ("naturally") in the electric field once CAs have been added to the gel, and where polymerization

efficiency is of little consequence. This discrepancy in

experimental ease is particularly acute for the presently

conventional mode of IPGEF involving the following steps: a)

Preparation of the light and heavy Immobiline mixtures; b) pH

neutralization of the Immobiline mixtures, c) gradient formation

by gradient mixer, d) polymerization at 50°C, e) washing, f)

drying to the original weight and g) pre-electrophoresis. A more

recent simplified procedure omits steps b, e, f, g) , and replaces

c) and d) by simpler procedures, replacing the gradient maker by

a pump and allowing for polymerization at room temperature or in

the cold (20). Although simplified, that procedure however does

not attain the degree of procedural simplicity achieved by IEF.

This difference may, however, cease to exist once commercially

available prefabricated IPGEF gels will be introduced.

6) Ease of pH gradient design:

One of the key advantages of IPGEF over IEF is the ease with

which the pH gradient can be computed on the basis of the known

pKs of the functional groups of Immobilines and of their volume

ratios (7,17). By contrast, since the chemical identities of the

23
CAs in IEF are unknown, pH gradients can only be modified by

trial-and-error to find a specific desired pH gradient. Even

when the CAs are replaced by simple buffers, which allows one to

compute steady-state pH gradients, realistic predictions seem hard to obtain: Those programs which neglect diffusion but are capable of rapidly and inexpensively estimating pH gradients with

up to 20 constituents provide insufficiently accurate predictions

(24) while those which consider diffusion require computer

simulation techniques and correspondingly more computing capacity

and time (25) which makes it economically prohibitive to predict

pH gradients with a practically useful number of 20 - 50 buffer

constituents

.

However, the predicted properties of pH gradients in IPGEF

also fail to depict experimental ones with sufficient accuracy:

At least in the wide (pH 4 to 10) pH range, the predicted even conductance across the pH gradient cannot be verified by

experiment (12). The discrepancy of experimental from predicted

pH gradients is less visible in view of the logarithmic nature of

pH. No data demonstrating statistically reproducible IPGEF pH

gradients are available as yet (as discussed above), and thus no rigorous testing of the accuracy of the predictions is possible.

Nonetheless, one can state a priori that the computation of pH

gradients neglects charge interactions between functional groups

which must exist in reality. The argument that only 1 in 50

residues carries a functional group, and that therefore charge

interactions are impossible, is fallacious since it neglects the

stereochemistry and the folding of polymer chains. Secondly,

inaccuracy must result from failure to incorporate 100% of

monomers into the polymer, in conjunction with the fact that the

% incorporation must vary among monomers with different

functional groups.

Notwithstanding all of these reservations, it remains clear

that simple predictability of pH gradients by computer simulation

is one of the real advantages of IPGEF over IEF.

24
7) Susceptibility to pH and voltage measurement:

Upon the introduction of IPGEF, it was taken for granted that in

view of the low ionic strength of the gel, pH could not be

directly determined by contact electrode. Instead, users of the

method have applied indirect ways to determine the pH gradient,

based on the steady-state positions of protein pi-markers.

However, the accuracy of such indirect methods of pH measurement

is difficult to ascertain since it depends on knowing the pi of

the markers at the temperature and ionic strength and the

viscosity of a gel, on their attainment of the steady-state as well as on the reagent purity and history of the protein markers.

Furthermore, the assumption of a curve shape between adjacent

markers and a curve defined by a small number of markers makes

this technique rather unreliable. Our laboratory succeeded in

taking direct pH measurements on IPG gels, using a relatively

wide (5 mm diameter) contact electrode, and by attaching a water

droplet to the electrode and a very long measurement time, up to

30 min at some pHs (20). This, of course, is not only

impracticably laborious but also inaccurate. The problem of

direct pH measurement was only solved in ICAPG gels where the

addition of CAs adds sufficient conductance to the gel to allow

for a rapid measurement by contact electrode, comparable to that

in IEF

.

It is concluded that direct pH gradient detrmination is one of

the key advantages of both IEF and ICAPG-EF over IPGEF.

In the practice of focusing, field strength measurement is far

less important than pH measurement. It becomes important in

trouble shooting, in addition to its theoretical interest. With

regard to field strength measurement, IEF, ICAPG-EF and IPGEF

present qualitatively the same problem of requiring a high

("infinite") impedance voltmeter. In all 3 cases, reliable

voltage measurement implies ascertaining that the field strength

25
values, added across the pH gradient gel, add up to give the

value of the applied potential across the gel; and that the

voltage profile across the gel be the same whether or not the

measurement proceeds progressively from either the anode or the

cathode (20)

.

8) Perturbation by gel hydration and dehydration phenomena:

Water extrusion from the gel during electrofocus ing is not a problem in tube or vertical slab apparatus, but presents a problem for horizontal slab gels in ICAPG-EF and, to a lesser

degree, in IPGEF. If the water exudates occur in the gel region

of interest, they will tend to produce diffuse zones. If they

are not, they appear harmless, and the effect can be neglected as long as the gel surface is wiped intermittently during

electrofocus ing

.

A second hydration-dehydration phenomenon of importance is the

formation of "ridges” on the gel surfaces of horizontal IEF and

ICAPG-EF gels. Presumably these "ridges" present osmotically

swollen zones of CAs . Although the "ridges" are visible in

horizontal slabs only, the must be equally present in vertical

slab or tube gels where they presumably give rise to the observed

irregular gel swelling along the gel length after gel removal

from the glass walls. In vertical apparatus, this differential

gel swelling along the pH gradient leads to a weakening of

adherence of the gel to the glass walls, with corresponding need

to strengthen wall adherence by coating of the glass with

polyacrylamide or agarose, by mechanically supporting the gel

(e.g. by Nylon mesh) and by its hydrostatic equilibration through

maintenance of equal liquid levels of anolyte and catholyte (26).

The "ridging" is not observed in IPGEF, except at concentrated

protein zones when the protein content is sufficient to cause the

osmotic adsorption of water into the zone. The extent to which "ridging" may affect protein patterns is

26
unknown; it may have a significant effect on the local field

strength. The relative prevalence of hydration problems appears

to be highest in ICAPG-EF, less in IEF and least in IPGEF.

9) Artefacts:

Very little evidence exists as to the degree of hydrophobic,

ionic or H-bond interactions between proteins and the charged

residues (CAs and Immobiline respectively) of either an IEF or an

IPG matrix. For IEF, all 3 types of interactions have been

demonstrated (27). Evidence also exists for ionic and H-bond

interactions in IPG gels (10). With regard to the former, IPG

should be more vulnerable than IEF in view of its lower ionic

strength. One would speculate, however, that hydrophobic

interactions should be less on IPG gels at least compared with

IEF gels in the alkaline range (28).

10) Compatibility with SDS-PAGE:

The coupling of EF with SDS-PAGE to give 2-D gels is one of

the important applications of EF. In that application, IEF gives

rise to interactions between basic and hydrophobic CAs, neutral

detergent (29) and SDS . The interaction products traverse and

perturb the 2nd dimensional SDS-gel by introducing into it a field asymmetry, Joule heating problems and different voltage

gradients and migration rates. These perturbations must be

expected to be absent in IPGEF, assuming that the IPG gel does

not contain any non-covalent ly adsorbed Immobiline or other

mobile ionized species. Nonetheless the transfer of protein

zones from IPGEF into the SDS-gel has been found to be inferior

to that from IEF (30). This may be due to a) electroendosmosic

interference with the implantation of the IPGEF-strip into the

SDS-matrix (22) , and/or b) to a greater insolubility of the zones which have approached the pi more closely, or have electrofocused

27
at the steady-state for a longer time, than in IEF (31). a) The

negative charging of the IPGEF-strip in a basic SDS-buffer seems responsible for producing a contact problem between strip and

SDS-matrix [Fig.l of (22)]. Transfer may also be impeded by

the cathodic direction of the electroendosmotic flow. The

contact problem appears largely remedied through an increase in

the viscosity of the IPGEF gel by the addition of glycerol to the

equilibration medium prior to strip application to the SDS-gel

(22). b) This interpretation is also consistent with the

beneficial effect of 30% glycerol in the IPG gel (22) since

protein insolubilization may be reduced in that medium. Such

interpretation may also be related to the higher potential

applied in IPGEF compared to IEF, causing a greater extent of

isoelectric precipitation.

One of the most important applications of the 2-D procedure

concerns the resolution of water- insoluble membrane proteins. In

that application, the EF gel has to contain dissociating agents

and detergents capable of maintaining the separated proteins in

solution; the most frequently used agents are 9 M urea, 2% NP-40

(alias Triton X-100) (32). For reasons of micellar size and

inhomogeneity, it appears preferable to exchange CHAPS or 3-octylglucoside for the NP-40 (33). The question then arises,

whether or not IEF and IPGEF are equally compatible with mixtures

such as 9 M urea, 0.01-0.05 M CHAPS. This should be the case in

view of the insensitivity of zwitterionic detergents to ionic

strength in the range of 0 to 0.2 M [Fig. 7 of (34)].

On balance, IPGEF and IEF appear to be both burdened with

problems upon coupling with a 2nd SDS -dimens ion , although the

nature of those problems is different. Weighing one set of

problems against the other, it appears impossible at this time to

judge which one is more perturbing to the 2nd dimensional gel

pattern.

11) Ease of protein isolation, preparative load capacity and

28
purity of product:

Preparative IEF can be conducted in a variety of gel media such

as Sephadex (dextran) , agarose, in addition to polyacrylamide, as well as in density gradients and in free solution, using various

types of apparatus. This versatility in preparative approaches

does not exist for IPGEF which is restricted to a polyacrylamide

matrix. However, IPGEF has the preparative advantage of avoiding

the presence of CAs in the fractionated zone and thus eliminates

the problems of separating them from the protein. Preparative

load capacity is mainly determined by 4 variables: a) The

cross-sectional area of the gel; b) the degree of distribution

overlap between the zone of interest and its nearest neighbors;

c) the water content of the gel (35); d) the limits imposed by

the buffering capacity of the CAs and/or Immobilines

respectively. In these regards, the sole preparative advantage

of IPGEF is that sample can be introduced to the limit posed by

item c), without consideration of focusing time and even in

continuous fashion, as pointed out in section 4. Furthermore, in

horizontal IPGEF gels the swelling of isoelectric protein zones creates a "water pocket" with enhanced capacity (36). To

accelerate preparative focusing, ICAPG-EF appears to be the

preferred method, unless an interaction between protein and CA

perturbs the resolution or product purity.

Subsequent to separation by either IEF or IPGEF, zones of

interest need to be excised, and protein needs to be freed of the

gel by either diffusion or electrophoresis. In both cases,

extraction of the protein by electrophoresis appears more

efficient. Numerous procedures and apparati for extraction in a continuous or discontinuous buffer system are available; the

latter has advantages with regard to product concentration (e.g.

37,38). A popular procedure embeds the slice to be extracted

into a horizontal gel and transfers the protein

electrophoretically into a trough containing a suitable adsorbent

29
(hydroxyapatite or ion exchange medium) (35). A very simple

isolation procedure which avoids zone excision in IPGEF has

recently been described, whereby the desired component is

collected within a trough cut in the gel at the position of its

pi; the trough is filled with suspension of Sephadex in water

(39).

Product impurity problems in IEF and IPGEF seem comparable

with regard to the polyacrylamide-derived non-proteinaceous

impurities which must be removed by gel filtration at 0°C in a subsequent step (40,41). Even IEF conducted in an agarose gel

gives off enough carbohydrate impurity to require such a final

purification step (ibid.). In view of this requirement, any

contamination of the product with CAs in either IEF or ICAPG-EF

becomes less important. But a quantitative removal of charged

small contaminants like the CAs or amino acids by gel filtation

is not possible - such removal requires an electrophoretic step.

It is concluded that in view of water accumulation in the

focused zone, and its tolerance for continuous or repetitive

sample application and consequent enrichment of the focused zone,

IPGEF has a greater load capacity than IEF. Quantitative data

concerning that advantage have been reported (36)

.

12) Equipment needs and cost:

Contrary to wide prejudice, the cost of Immobilines per EF

separation is equivalent to that of CAs. However, IPGEF is

undoubtedly the more expensive method in view of the need for a gradient former and a high-voltage power supply capable of

delivering at least 100 V/cm of gel, and preferably as much as 500 V/cm. Since either 2-chamber gradient mixers, or proportioning pumps (20) are commonly available in biochemical

laboratories, the need for a gradient forming device can be

discounted. For constructing specific non-linear pH gradients,

specialized apparatus is required (9). With the development of

30
improved Joule heat dissipation techniques, such as the

introduction of horizontal slab apparatus cooled by Peltier cells

(e.g. Isobox, Hoefer Instruments), high field strength will

become a routine way for increasing resolution in other forms of

gel electrophoresis as well, so that the need for a 5,000 to

10,000 V power supply in IPGEF will become increasingly

non-specific

.

The only other equipment - pH contact electrode, infinite

impedance voltmeter and micro-ammeter - is also equally needed

for IEF and IPGEF.

It is concluded that equipment needs for IPGEF are greater

than those for IEF. This difference may disappear with time.

References

1) Gasparic, V. , Bjellqvist, B. and Rosengren, A. (1975) Swedish

Patent No. 14049-1.

2) Rilbe, H. , in: Catsimpoolas , N. (Ed.), Isoelectric Focusing,

Academic Press, New York NY 1976, pp. 14-52.

3) Fawcett, J. S. and Chrambach, A., Electrophoresis 1986, in

1 press

4) Caspers, M. L. , Posey, Y. and Brown, R. K. , Anal. Biochem.

1977, 79, 166-180.

5) Chrambach, A. and Nguyen, N. Y. , in: Catsimpoolas, N. (Ed.)

Electrophoresis '78, Elsevier, North Holland Publ. Co.,

Amsterdam-New York NY 1978, pp . 3-18.

6) An der Lan, B. and Chrambach, A., Electrophoresis 1980, 1 , 23 - 27 .

31
7) Gianazza, E., Astrua-Testori , S. and Righetti, P. G.,

Electrophoresis 1985, 6, 113-117.

8) Goerg, A., Postel, W., Weser, J. , Patutschnick, W. and Cleve,

H., Am. J. Hum. Genet. 1985, 37, 922-930.

9) Altland, K. , Hackler, R. , Banzhoff, A. and v. Eckardstein, A.,

Electrophoresis 1985, 6, 140-142.

10) Righetti, P. G. , J. Chromatography 1984, 300 , 165-223.

11) Allen, R. C., Electrophoresis 1980, 1, 32-36.

12) Fawcett, J. S. and Chrambach, A., Electrophoresis 1986,

submitted (The Voltage Across Wide pH Range IPG Gels and Its

Modulation Through the Addition of Carrier Ampholytes). ^

13) Murel, A., Vilde, S., Kongas , A. and Kirret, 0. 1984,

Electrophoresis 5, 139-142.

14) Jackiw, B. A. and Brown, R. K. , Electrophoresis 1980, 1 , 107-112.

15) Voris, B. P. and Young, D. A., Anal. Biochem. 1980, 104 , 478-484.

16) Baumann, G. and Chrambach, A., in: Righetti, P. G. (Ed.)

Progress in Isoelectric Focusing and Isotachophores is , Elsevier,

Excerpta Medica, North Holland, Assoc. Sci. Publ., Amsterdam,

The Netherlands 1975, pp . 13-23.

17) Altland, K. and Altland, A., Clin. Chem. 1984, 30,

2098-2103.

32
18) Pietta, P., Pocaterra, E., Fiorino, A., Gianazza, E. and

Righetti, P. G. , Electrophoresis 1985, 6, 162-170.

19) Goerg, A., Westermeier, R., Postel, W., Gianazza, E. and

Righetti, P. J., J. Biochem. Biophys. Methods 1980, 3, 273-284

20) Fawcett, J. S. and Chrambach, . A. , Electrophoresis 1986,

submitted (Simplified Procedure for the Preparation of IPG-Gels)

1

21) Righetti, P. G. , Ek, K. , and Bjellqvist, B. 1984, J.

Chromatogr. 1984, 291 , 31-42.

22) Goerg, A., Postel, W., Guenther, S. and Weser, J.

,

Electrophoresis 1985, 6, 599-604.

23) Rimpalainen, M. H and Righetti, P. G. , Electrophoresis 1985,

6, 419-422.

24) Chrambach, A., in: Neuhoff, V. (Ed.) Electrophoresis '84

Verlag Chemie, Weinheim FRG, pp. 3-29.

25) Mosher, R. A. and Thormann, W. , Electrophoresis 1985, 6,

477-482.

26) Nguyen, N. Y. , McCormick, A. G. and Chrambach, A., Anal.

Biochem. 1978, 88, 186-195.

27) Cuono, C. B., Chapo, G. A., Chrambach, A. and Hjelmeland, L.

M., Electrophoresis 1983, 4, 404-407.

28) Righetti, P. G., Gianazza, E., Brenna, 0. and Galante, E., J

Chromatogr. 1977, 137 , 171-181.

33
29)

Gianazza, E., Astorri, C. and Righetti, P. G. , J. Chromatogr.

1979, m, 161-169.

30) Dunn, M. J. and Patel, K. , in: Radola, B. J. (Ed.)

Elektrophorese Forum '85 Technische Universitaet Muenchen,

Muenchen, FRG, pp. 45-54.

31) Poehling, H. M. and Neuhoff, V. , Electrophoresis 1980, 1 , 90-102.

32) O'Farrell, P. H. , J. Biol. Chem. 1975, 250, 4007-4021.

33) Hjelmeland, L. M. and Chrambach A., in: Jakoby, W. B. (Ed.)

Methods in Enzymology Vol. 104, Academic Press, New York NY 1984,

pp. 305-318.

34) Hjelmeland, L. M., Nebert, D. W. and Chrambach, A., in:

Catsimpoolas , N. (Ed.) Electrophoresis '78, Elsevier, North

Holland Publ. Co., Amsterdam-New York NY 1978, pp. 29-56.

35) Casero, P., Gelfi, C. and Righetti, P. G., Electrophoresis

1985, 6, 59-68.

36) Righetti, P. G. and Gelfi, C., J. Biochem. Biophys. Methods

1984, 9, 103-119.

37) Hjerten, S., Liu, Z.-Q. and Zhao, S.-L., J. Biochem.

Biophys. Methods 1983, 7_, 101-114.

38) An der Lan, B., Horuk, R. , Sullivan, J. V. and Chrambach, A.,

Electrophoresis 1983, 4, 335-337.

39) Bartels, R. and Bock, L. , in: Neuhoff, V. (Ed.),

Electrophoresis '84, Verlag Chemie, Weinheim FRG, pp. 103-106.

34
40) Chrambach, A. and Rodbard, D. , in: Hames , B. D. and Rickwood

D. (Eds.) Gel Electrophoresis of Proteins: A Practical Approach,

Information Retrieval Ltd. Press, London and Washington DC 1981,

pp. 93-143.

41) Chrambach, A., in: Neuhoff, V. and Maelicke, A. (Eds.) The

Practice of Quantitative Gel Electrophoresis, Verlag Chemie,

Weinheim FRG 1985, pp. 1-265.

Footnote

Available upon request

Acknow 1 edgement

We thank Dr. Dietmar Tietz for entering the text into the IBM

6670 printing system.

35
Carl R. Merri 1 , M. D.

Section on Biochemical Genetics

Clinical Neurogenetics Branch

National Institute of Mental Health

Bethesda, Maryland 20892

Stains tor the

Detection of Proteins

Separated by

Electrophoretic Methods

The development of electrophoretic techniques has been

paralleled by development of Protein detection methods with

ever-increasing sensitivities. Detection techniques have

progressed from direct observation of protein-coated

micro spheres and colored proteins, requiring milligram amounts,

to the detection of proteins and by their absorption of

ultraviolet light, the observation of schlieren patterns,

labelling with radionuclides, by direct staining with organic,

fluorescent dyes, and most recently by silver stains which

require a tenth of a nanogram. Many detection techniques may be

employed quantitatively, provided that their methodological

limitations are respected. Most protein stains and

autoradiographic methods exhibit protein-specific quantitative

optical den sit '//concentration relationships, which are indicative of the dependence of detection methods on the

content of specific groups within each protein. These

protein-specific staining relationships may be utilized to

differentiate proteins, and emphasize the need to limit

quantitative comparisons to homologous proteins. So long as intergel studies are confined to comparisons of homologous

proteins and observations are made within the nonsaturation

range of the detection procedures, valid quantitative results

may be obtained.

1. Historical Introduction

The observations in 1807, that colloidal particles migrate in electrical

fields, by the physicist Ferdinand Frederic Reuss, initiated the

development electrophoretic separation techniques C 1 3 . Applications of

electrophoretic methods to problems of protein and nucleic acid

purification would not have been possible without complementary detection

methods. The earlist applications of electrophoresis relied on direct

observations of objects including, cells, colloidal particles, bacteria,

and the naturally colored proteins such as, myoglobin, hemoglobin,

ferritin, and cytochrome c C 2 3 . However, most protein molecules could not

be observed directly with visible light and their el ectr ophor et i c properties were studied by observing quartz microspheres with the proteins

36
adsorbed to their sur-face [3-5].

Detection o-f non-colored proteins by the specific absorption of ultraviolet

light was first demonstrated by Tiselius in the 1930s [61. Despite its

early introduction, protein detection by ultraviolet absorption has never gained widespread acceptance, primarily because it requires a special light

source, filters, and optical components that are transparent to ultraviolet

light. Tiselius also utilised schlieren, or shadows, created by boundaries

between regions with different refractive indices due to the varing

concentrations of proteins in electrophoretic systems as a detection method

[61. This system also required complex optical systems.

Introduction of organic stains for detection of proteins and eliminated

many of the complications inherent in the earlier systems and often

provided increased sensitivities. The use of moist filter paper as an electrophoretic support medium or carrier for zonal electrophoretic

separation stimulated the adaptation of a number of histochemical stains

for the detection of uncolored proteins [73. These stains were usually

employed after the proteins were "fixed" or made immobile, to decrease

protein loss in the staining solutions. Heat, 110°C after electrophoresis

was one of the first methods used to "fix" proteins. Early organic protein

stains included Bromophenol Blue [8] and Amido Black [93. Lipoproteins were preferentially stained by Oil Red 0 [103, while glycoproteins were detected

by a red color that was produced upon their oxidation with periodic acid

and subsequent reaction with fuchsin sulfurous acid (Schiff's reagent)

[113. Coomassie Blue stains, with their capability of detecting as little

as half a microgram of protein, are the most sensitive of these organic

protein stain [123. This increased sensitivity of the Commassie Blue stains

was originally used to detect proteins separated on cellulose acetate. It was also complimented the increased protein resolution of acrylamide gel

electrophoretic methods. Most of the early organic stains were employed to

detect proteins after their electrophoretic separation,

post -electrophoretic stains. Fluorescent protein stains which were introduced by Talbot and Yaphantis in 1971 [133 can now detect as little as one nanogram of protein [143. However, these fluorescent stains usually

require reaction conditions that are best performed prior to

37
si ectrophor esi s , pr e-el ectr ophor et i c stains, and their formation of

covalent bonds with the protein molecules generally alters the charge of

the proteins [15]. This charge alteration is not of consequence for

electrophoretic techniques that separate proteins on the basis of molecular

weight, such as sodium dodecyl sulfate (SDS) el ectrophor esi s , but it can alter separations by isoelectrofocusing [153.

Radioactively labelled proteins may be visuallized without staining by

autoradiographic methods which were first introduced by Becquerel and Curie

in their discovery of the phenomenon of radioactivity [163, or by

fluorographic techniques which were introduced by Wilson to study tritium

labelled metabolites involved in photosynthesis [173. Proteins

radioactively labled to a high specific activity can be detected with

sensitivities equal to, and often better, than those obtained by the most

stains. However, the use of radioactively labled proteins is limited, as it

is difficult to achieve high specific activities in animal studies and

unethical in reseach involving humans.

Introduction of silver as a general protein stain increased the sensitivity

of protein detection to a tenth of a nanogram [18-193. This was a 100 fold

gain over that attained by the most sensitive commonly used organic stain,

Coomassie Blue. The first silver stains used, for detection of proteins

separated by polyacrylamide gel electrophoresis were adapted from

histological silver stains. These were often tedious, requiring three hours

of manipulations and use of numerous solutions [18-193. In the seven years

since the introduction of silver staining as a general method for the

detection of proteins in polyacrylamide gel electrophoresis numerous staining proptocols have been developed [203. These protocols can be

divided into three categories: the diamine or ammoniacal silver stains, the

non-diamine stains including stains based on photographic chemistry, and

stains based on the photo-development or photo-reduction of silver ions to

form the metallic silver image. Some of the newer silver stains can be

performed in less than fifteen minutes and they may be employed for

quantitative analyses.

2 Post-Electrophoretic Organic Protein Stains

38
2.1 Commassie Blue stains

C d o iri a s s i e Blue stains are the most comm only used organic stains for

post-electrophoretic detection of proteins separated on polyacrylamide

gels. They were originally developed as acid wool dyes and they were named

"Coomassie dyes" to commemorate the 1896 British occupation of the Ashanti

capital, Kumasi or "Coomassie", now in Ghana. In a collaborative effort to

find a highly sensitive protein stain that could be used in quantitative

studies, a microbiologist of the Australian National University, Faze k as de

St. Broth, and textile chemists at the University of New South Wales School

of Textile Technology (Australia) tested numerous dyes and dyeing

techniques [211. Their studies demonstrated the intense protein staining

abilities of the triphe.nylmethane Coomassie stains.

Coomassie Brilliant Blue R250 (the letter "R" stands for a reddish hue

while the number "250" is a dye strength indicator) was the first of the

tr i phenyl methane stains to be introduced. It can detect as little as

0. 5ug/cm of protein and gives a linear response up to 20ug/cm. However, it

should be noted that the relationship between stain density and protein

concentrations varied for each of four proteins tested [213. Fazekas de St.

Sroth et al. originally introduced this stain to detect proteins on cellulose acetate, agar or starch gels C 1 2] . Meyer and Lamberts (1965)

adapted the Coomassie Brilliant Blue R250 stain for polyacrylamide gels

[213. They used electrophoresis to remove excess stain, but a number of

proteins displayed some mobility during this destaining [21-233.

Non -electrophoretic methods of destaining have largly eliminated this

problem. Other Coomassie stains, such as Coomassie Brilliant Blue G250 ("G"

indicates that this stain has a greenish hue), have augmented the stain

introduced by Fazekas de St. Groth et al.. Coomassie Brilliant Blue G250

has a diminished solubility in 127. TCA. permiting its use as a colloidal

dispersion. Such a colloidally dispersed dye does not penetrate gels,

permitting rapid staining of proteins without an undesired background [243.

Another derived Coomassie stain, Coomassie Violet R 1 5 0 , has gained some

favor by virtue of its ability to rapidly stain proteins on polyacrylamide

gels and its ease of destaining [25-263. Although Coomassie Violet R150 is

39
no longer produced bv Imperial Chemical Industries, Ltd., a very similar

dye, Serve Violet 49, is available. Serva Violet 49 differs from Coomassie

Violet 150 by the substitution of a d i ethy 1 ami ne group for a d i methyl ami ne group. As Imperial Chemical Industries, Ltd. no longer produces Coomassie

Blue R250 or G250. They still hold the trademark "Coomassie", so that

manuf acturers who are currently producing Coomassie type dyes have had to

introduced their own trademarked names [27].

2.2 Mechanism of Coomassie Brilliant Blue Staining

Coomassie Blue staining requires an acidic medium for electrostatic

attraction to form between the dye molecules and the amino groups of the

proteins. This ionic attraction, together with van der Waals' forces,

binds the dye-protein complex together. The binding, however, is fully

reversible by dilution under appropriate conditions [123. The relatively

high staining intensity of Coomassie Blue stains, compared to other organic

dyes , is apparently due to secondary bonds formed between dye molecules.

Additional dye may be bound by dye-dye interactions to dye molecules that are ionically bound to, or in hydrophobic association with, protein

molecules [281.

Recent studies concerning the mechanisms of Coomassie dye staining of

proteins have indicated the importance for the basic amino acids. Righetti

and Chillemi noted that polypeptides rich in lysine and arginine were aggregated by Coomassie G dye molecules, suggesting that the dye interacts

with the basic groups in the polypeptides [29]. Studies of proteins with

known sequences, by Tal et a 1 . , have confirmed these observations and

dem on s t rationed of a significant correlation between the intensity of

Coomassie blue staining and the number of lysine, histidine and arginine

residues in the protein [30].

f 2.3 Metachromatic effects

Secondary dye-binding or dye-dye interactions may play a fundamental role

in the metachromatic effects that have been observed with some polypeptides

that are stained with Coomassie Brilliant Blue R250 [31-321. Some

40
peripheral nerve proteins stain to produce both red and blue bands with

Coomassie Brilliant Blue R 2 5 0 after separation by SDS-pcly acrylamide

electrophoresis. Cal-f skin collagen and histone-1 protein display similar

red-staining bands, as do most other collagen samples [32]. Metachromatic

shifts are often observed when dye molecules are stacked together [33].

Such dve stacking or close aggregation may affect the dye's electron

resonance structure, resulting in an altered response to excitation by

light.

These metachromatic shifts are affected by a number of variables. Bands

containing more than 5 ug of a protein that normally produces a red hue

have been shown initially to stain blue in the center of the band. Only

with continued destaining does the red become apparent. Red-staining

proteins such as histone-l or collagen may be converted to blue bands by

placing the gels in solutions containing alcohol, SDS or TCA [32].

Met achr cmasy is also diminished by staining at elevated temperatures [31].

The dependence of metachromic effects on protein specificity,

concentration, temperature, solvents, and the type of spectral shift

observed with Coomassie Blue are characteristic of metachromatic mechanism

rather than staining artifacts due to dye contaminants.

2.4 Other Common Post-El ectr ophoreti c Organic Protein Stains

Ami do Black (Acid Black 1) and Fast Green (Food Green 3) are commonly

utilized for protein detection following polyacrylamide gel

electrophoresis. However, Coomassie Blue R250 staining is three times the

intensity of Fast Green and six times the intensity of Amido Black staining

[271. The staining intensities of these dyes are approximately proportional

to their relative molar adsorption coefficients. Wilson [28] estimated

that 1 mg of protein will bind approximately 0.17 mg of Amido Black, 0.23

mg of Fast Green, 1.2 mg of Coomassie Blue R250 and 1.4 mg of Coomassie

Blue G250. It is unlikely that this variation is due to variations in

molecular weight between these dyes since their weights only range between

616.5 daltons for Amido Black and 854.0 daltons for Coomassie Blue G250.

Variations in binding must be due to an differences in the number of dye

molecules bound per protein molecule. The higher staining intensity

41
Coomassie Blue G 2 5 0 may be due to this dyes higher efficiency at forming

dyp-dye interactions or hydrophobic interactions between the dye molecules

and the proteins. Secondary binding mechanisms also occur with Amido Black

and Fast Green dyes, although perhaps not at levels observed with Coomassie

Blue R250, as these dyes display met a chromic effects with certain proteins

similar to the metachromasy observed with Coomassie Blue R250. Amido Black

produces blue-green bands with certain histones rather than its

characteristic blue-black color, while Fast Breen produces a difference in

the ratio of blue to green hues C 3 4 ]

.

3 Pr e-El ectr ophor eti c Organic Protein stains

P re-electrophoretic organic stains usually involves the covalent binding of

either a fluorescent or colored residue to the protein prior to

electrophoresis. Potential advantages of these stains include: the

possibility of performing stoichiometric reactions with proteins without

the diffusion limitations imposed by staining within a gel matrix, the

process of electrophoresis can be followed visually with "stained''

proteins, and there are no background problems due to dye-trapping or reaction of the dye with the gel. An often cited disadvantage of covalently

bound pre-elect r op horetic stains is that they usually alter the charge of

proteins E 1 5 ] . This objection is not of consequence for sodium dodecyl

sulfate (SDS) electrophoresis, as the protein's mobility depends on molecular weight, and the dye molecules are usually too small to produce an appreciable effect. Furthermore, as long as the stains react with proteins

in a stoichiometric manner, shifts in protein pattern should be highly

reproducible, permitting construction of valid protein maps and protein

identifications.

3.1 Non-f 1 uor escent Stains

Remazol Brilliant Blue R, was the first anionic dye used for prestaining

proteins [353. A major disadvantage was its lower limit of sensitivity (3ug

of protein). Boss hard and Datyner introduced anionic dyes with

sensitivities comparable to the Coomassie Blue stains, Drimarene Brilliant

42
Blue K-BL and Uniblue A [15]. These dyes are capable of detecting bands

containing as little as 0.5 ug o-f protein. They react primarily with the

amino groups of proteins as well as the hydroxyl groups of serine and

tyrosine. A similar range and sensitivity has been achieved with a cationic dye that binds to the protein's amino groups, causing less

alteration in isolectric focussing patterns than anionic dyes [36].

3.2 Fluorescent stains

Fluorescent stains are currently the most sensitive pre-electrophoretic

stains in use. The first fluorescent stain used to visualize proteins in

gels was anil i nonaphthalene sulfonate, a post-electrophoretic stain which

is thought to bind to a protein's hydrophobic sites, to form a fluorescent

complex [37]. Its limit of sensitivity is about 20 ug of protein.

Pre-electrophoretic fluorescent staining with dansyl chloride was first

introduced by Talbot and Vaphantis [13]. This stain reacts with proteins to

form fluorescent derivatives in 1-2 minutes at 1 0

0

° C , with a sensitivity

limit of 3-10 n g

.

Reagents that were first designed to increase the detection limits of amino

acid analyzers have resulted in a number of highly sensitive flourescent

stains [38]. The first of these, f 1 uor escami ne , is a non-f 1 uor escent

compound however at room temperature and alkaline pH, it reacts with

primary amines of amino acids within to yield a fluorescent derivative. It has proven capable of detecting as little as 6 ng of myoglobin [39-40]. A related compound, 2-methoxy -2, 4-diphenyl- 3 (2H)-furanone (MDPF ) has the same speed and simpicity of reaction as fluorescamine, and its protein

derivative is 2.5 times as fluorescent as a f 1 uor escami ne-1 abel 1 ed protein.

Furthermore it does not fade as rapidly. As little as 1 ng of protein has

been detected with MDPF. It also has a linear response from 1-500 ng. As

with most other protein stains, a plot of relative fluorescence versus protein concentration revealed a different slope for each of four proteins

studied [14].

Although these fluorescent stains achieve greater sensitivity than other

organic stains, they require ultraviolet light for visualization, and

43
direct quantitation requires -fairly sophisticated equipment. These

problems, coupled with the altered electrophoretic mobility during

isolectric focussing (a result of the altered protein charge(s) from the

fluorescent derivatization), has tended to inhibit utilization of these

fluorescent stains.

€

4 Silver Stains

Silver nitrate, the main ingredient in silver stains, was first described

by the Arabian alchemist, Gabir Dschabir ibn Hajjam in the eighth century.

The observation that this compound has the ability to blacken when in

contact with organic substances, including human skin, is usually credited

to Count Albert von Bollstadt [411. Modern applications of this property of

silver nitrate began with Krause's 1844 observation that silver nitrate

solutions could be used to stain small pieces of fresh tissue [421. Golgi

and Cajal used silver stains to establish the foundations of modern

neuroanatomy [431. Cajal also adapted photochemical methods to develop new histological silver stains that produced better definition of nerve fibers.

In general, since the time of Cajal, histological silver stains have become

more complex as empirical alterations have been made in the procedures to

increase the specificity of the staining for specific cells or subcellular

structures.

Introduction of silver as a general stain for proteins separated by

polyacrylamide gel electrophoresis permitted the detection of as little as 0.1 n g of protein. This silver stain, an adaptation of a histological

silver stain, was demonstrated to be 100-fold more sensitive than previous

stains [16-19], Other histological silver stains adapted for protein

detection were limited to the detection of specific proteins, modifying

subgroups, or to proteins electrophoresed in agarose. A modified Gomon

silver stain was used to study phosphorylase [45], while another

histological silver stain, originally developed to visualize nucleoli, was used to detect nucleolar proteins. Kerenyi and Galiyas adapted a histological silver stain to visualize cerebrospinal fluid proteins

separated on agarose [47-48]. This stain did not achieve widespread

acceptance, perhaps because it produced numerous staining artifacts, did

44
not work well in polyacrylamide, or because of a report that it was quantitatively irreproducible [493. However , recent work by has improved

this stain's performance in agarose [501.

In the seven years since the introduction of silver staining as a general

method for the detection of proteins sparated by polyaccrylamide gel

electrophoresis numerous staining protocols have been developed. These

protocols can be divided into three basic catogories: the diamine or

a m m o n i a c a 1 silver stains, the non -diamine chemical development silver

stains, and the silver stains that depend on light for the photoreduction

of the silver ions to form the metallic image.

4.1 Diamine Silver Stains

These stains rely on the stabilization of the silver ions by the formation

of silver diamine complexes, with ammonium hydroxide. Diamine silver stains

were first developed for the visualization nerve fibers C 5 1 3 . Silver ion

concentrations are usually very low in these stains, as most of the silver

is bound in diamine complexes [52]. Diamine stains tend to become

selectively sensitive for glycoproteins if their concentration of silver

ions is decreased. This specificity can be minimized by maintaining a sufficient sodium to ammonium ion ratio in the diamine solution [53].

However, in some applications, an emphasis of the diamine stains

specificity has proven useful, as in the adaptation of a diamine

histological silver stain to visualize neurofilament polypeptides in

electrophoretic analyses of spinal cord homogenates [54].

In the diamine stains, the ammoniacal silver solution must be acidified,

usually with citric acid, for image production to occur. The addition of

citric acid lowers in the concentration of free ammonium ions, thereby

liberating silver ions to a level where their reduction by formaldehyde to

metallic silver is possible. The optimal concentration of citric acid also

results in a controlled rate of silver ion reduction, preventing a non -selective deposition of silver.

4.2 Non-Diamine Chemical Development Silver Stains

45
Host of the non-die mine chemical development silver stains were developed

by adapting photographic photochemical protocols [55-5?]. These stains rely

on the reaction of silver nitrate with protein sites in acidic conditions,

followed by the selective reduction of ionic silver by formaldehyde in

alkaline conditions. Sodium carbonate and/or hydroxide and other bases are used to maintain an alkaline pH during development. Formic acid, produced

by the oxidation of formaldehyde, is buffered by the sodium carbonate [603.

4.3 Photo-development Silver Stains

Photo -development stains utilize energy from photons of light to reduce

ionic to metallic silver. Scheele in 1777 recognized that the blackening of

silver chloride crystals, by light was due to the formation of metallic

silver [613. This ability of light to reduce ionic to metallic silver was adapted by William Fox Talbot, in 1839, as the basis of a photographic

processes that dominated photography from its introduction until 1862, when

photo-development was replaced by "chemical development" processes [623.

The use of photo-reduction provides for a rapid, simple, sensitive silver

stain method for detecting proteins [63-643.

Most chemical development stains require a minimum of two solutions, in

addition to the fixing solution. This requirement for multiple solutions is

a result of the use of alkaline solutions for the chemical reduction of

silver. The presence of silver ions and an organic reducing agent in an alkaline solution often results in the uncontrolled reduction of silver.

However, since light can reduce silver in an acidic solution, a photo- development stain may utilize a fixation solution followed by a single staining solution. Such single-solution photo-development silver

stains have two major advantages over chemical-development silver stains.

First, pH gradient effects are eliminated. In chemical development, one solution, containing silver ions, diffuses out of the gel, while the

solution containing the reducing agent diffuses into the gel. The

interactions of these solutions creates complex pH gradients within the

gel. A single-solution photo-development stain reduces such diffusion

effects, minimizing staining artifacts due to variations in gel thickness.

46
Proteins on ultra-thin supporting membranes such as cellulose nitrate stain

poorly with the "chemical stains" because they retain very little silver

nitrate when transferred into alkaline solution for image development.

Because the photo- development stain contains the silver ions in the

image-developing solution, proteins may be visualized even when bound to

thin membranes. *

4.4 Combinations of Photo-Development And Chemi cal -Devel opment Stain

By combining photo-development and chemical-development methods, a stain

has been developed which can detect proteins and nucleic acids in the

nanogram range, it can be performed in under fifteen minutes, and it

results in minimal background staining [651. This stain utilizes: a silver

halide, to provide a light senstive detection medium, and to prevent the

loss of silver ions from membranes or thin layer plates; photo-reduction,

to initiate the formation of silver nucleation centers; and

chemical-development, to provide a high degree of sensitivity by depositing

additional silver on the silver nucleation centers (formed by the

photo-reduction of the silver halide). This stain displays an average

detection sensitivity of 1 ng of protein or lOng of DNA. The stain's

rapidity of action, and its' ablility to stain samples spotted on membranes, such as cellulose nitrate, has afforded the opportunities to

investigate aspects of the mechanism of silver staining.

The first step in this stain protocol employs copper acetate, a metal salt

that is both a good fixative [66] and a silver stain enhancer. The

mechanism of copper's stain enhancement, in this and other silver stains,

may be similar to its action in the biuret reaction [19], in which a characteristic color shift, from violet to pink, is achieved by titrating

peptides in the presence of capper ions. Copper complexes formed with the

N -peptide atoms of the peptide bonds are primarily responsible for this

reaction. There are also some number of secondary sites which may interact

with copper. Any elemental copper formed may displace positive silver ions

from solution as copper has a greater tendency to donate electrons than

silver, indicated by its position in the electromotive series of the

elements. Following the treatment with copper acetate, the membrane is

47
sequentially soaked in a solution containing chloride and citrate ions and

then in a solution containing silver nitrate. The membrane is then

irradiated with light while it is in the silver nitrate solution. The

presence o f the resulting silver chloride, in the membrane, produces a significant increase in light sensitivity over that which can be achieved

with silver nitrate alone. Herman Vogel, a 19th century photochemist,

suggested that although silver chloride is more sensitive to the reducing

action of light than silver nitrate, it is fixed in position by its

insolubility and the potential density of its image would be limited unless

the free silver ions supplied by the silver nitrate are present to diffuse

into the photo-reduction centers [671. This increase in sensitivity was further enhanced in this stain by the presence of acetate and citrate ions

[64,67-681. White fluorescent light proved to be the most effective for

this photo-reduction. Ultra violet light produced a denser image, but it

also produced an unacceptable background stain. Continued irradiation with

white light would provide sufficient photo-reduction to produce an image of

the protein pattern on the membrane, however, photo-reduction alone usually

results in a dense background stain when applied to thin membranes [641. By

limiting the light irradiation to a total of four minutes, only enough to

initiate the formation of a latent image, formation of a visiable image is

achieved by chemical-reduction. The chemical-reduction of ionic to metallic

silver was effected by placing the membrane in a solution containing the

reducing reagents hydroquinone and formaldehyde. During image formation,

ionic silver is reduced to metallic silver, formaldehyde is converted to

formic acid [601 and hydroquinone to quinone. Unreacted silver chloride is

removed from the membrane, to prevent a grayish cast background, and

continued darkening of the membrane as the silver ions in the unreacted

silver chloride are photo-reduced, by exposure to light. Removal of the

silver chloride is accomplished by complex ing the silver chloride with

sodium thiosulfate to form a series of complex argentothiosulfate sodium

salts, most of which are soluble in water [621. The argentothiosulfate

sodium salts, unreacted reagents, and silver grains formed in solution that

may have precipitated onto the surface of the membrane are washed away with

water.

48
4.5 General Silver Stain Mechanisms

The basic mechanism underlying all protein detection silver stains involves

reduction of ionic to metallic silver. Detection of proteins in the gel or membrane requires a difference in the oxidation-reduction potential between

the sites occupied by proteins and adjacent sites of the gel or membrane.

If a protein site has a higher reducing potential than the surrounding gel

or matrix, then the protein will be positively stained. Conversely, if the

protein site has a lower reducing potential than the surrounding gel or matrix, the protein will appear to be negatively stained. These relative

oxidation-reduction potentials can be altered by the chemistry of the

staining procedure. Proteins separated on polyacrylamide gels have been

shown to stain negatively if the gel is soaked in the dark in silver

nitrate followed by image development in an alkaline reducing solution

(such as Kodak D76 photographic developer). By treating the gel with

potassium di chromate prior to the silver nitrate incubation followed by

development of the image in an alkaline reducing solution (utilising

formaldehyde as the reducing agent), a positive image is produced C53].

Positive images may also by obtained by substituting potassium ferric yanide

[ 54 ], potassium permanganate C 6 9 ] , or di thi othrei tol C59] for the potassium

dichromate in this stain. Dichromate, permanganate and ferric yanide are thought to enhance the formation of a positive image by converting the

protein's hydroxyl and sulfhydryl groups to aldehydes and thiosulfates,

thereby altering the oxidation-reduction potential of the protein. Although

the formation or presence of aldehydes has often been suggested as essential for silver staining, in certain histological stains neither

aldehyde-creating or aldehyde-blocking reagents appreciably affect silver

staining [701. Dithiothreitol, a reducing agent, also creates a positive

image, perhaps by maintaining the proteins in a reduced state. However,

other reducing agents, such as beta-mer captoethanol , do not enhance

positive image formation. Alternatively, all of the positive image

enhancing compounds may form complexes with the proteins and these

complexes may act as nucleation centers for silver reduction [20].

49
4.6 Reactive Groups on Proteins which affect Silver Stains

The combination photo-development, chemical-development silver stain has

been utilized to study amino acid homopolymers and individual amino acids

to gain information about reactive groups that may be involved i n silver

staining reactions [653. The only individual amino acids which stained were cysteine and cystine. Poly -methionine and the hydrophilic basic amino acid

polymers: poly-lysine, poly -arginine, poly -histidine, and poly -ornithine

also stained E 65 3 . Staining of the basic amino acids in their homopolymeric

form, but not as individual amino acids, may be related to the shift of p K s that is normally associated with the incorporation of amino acids into

peptides. This shift in pK toward the neutral range results in an increased

presence of ionised amino acid side chains closer to the physiological pH.

The ability of reactive group in an amino acid side chains to form

complexes with metal ions may be enhanced by such a shift. For example, a shift in the p k of an amino group would reduce the proton competetion that

a metal ion must overcome for the amino group's N-atom electron pair.

Staining of the basic amino acid and methionine homopolymers, but not their

individual amino acids may also indicate the need for cooperative effects

of several intramolecular functional groups to form complexes with the

silver or copper ions E 7 1 3

.

Heukeshoven and Dernick also observed silver staining of the basic

homopolymers of histidine, arginine, and ornithine, although they did not

report staining of the basic amino acid homopolymer poly-lysine [723.

Furthermore Nielsen and Brown noted that the basic amino acids: lysine,

arginine, and histidine, (in both a free and homopolymeric form) produced

colored complexes with silver [733.

Heukeshoven and Dernick reported silver staining of the homopolymers of

glycine, serine, proline and aspartic acid [723 while Nielsen and Brown

reported the formation of colored silver complexes with: aspartate, and

tyrosine [733. Staining of these homopolymers was not observed in the study

of Nerril and Pratt [653. In this regard prior metal binding studies have

failed to demonstrate metal interactions with the side-chain hydroxyl

groups of serine, threonine or tyrosine [713. These discrepancies

50
concerning the n c r. - b a s i c amino acids may be due to differences in the

staining procedures employed; the H e u k e s h o v e n and D e r n i c k study stained

homopolymers on polyacrylamide gel, while Nielsen and Brown studied

formation of silver-amino acid complexes in solution. Both of these studies

used formaldehydye in an alkaline sodium carbonate solution for image

development, while Merril and Pratt utilized acidic conditions and a combination of light, hydroquinone and formaldehyde for image formation

[65]

.

The importance of the basic and the sulfur containing amino acids has been

corroborated by observations with purified peptides and proteins of known

amino acid sequence [651. Leucine enkephalin, which has neither sulfur

containing nor basic amino ac i d s does not stain with silver, while

neurotensin which also has no sulfur containing amino acids but does have

three basic amino acid residues (one lysine and two arginines) does stain.

Gastrin produced a weak staining reaction. It lacks basic amino acids but

it has one sulfur containing amino acid, methionine. Oxytocin stains fairly

vigorously. It also has no basic amino . acids but it does have two sulfur

containing cysteines. The staining reaction of angiotensin II was rather

anomalous. It produced a negative stain rather than a positive silver stain

despite its two basic amino acids, arginine and histidine. All the other

polypeptides; insulin somatostatin, alpha-melanocyte stimulating hormone,

thyrocal ci to.ni n , aprotinin, vasoactive intestinal peptide and ACTH,

contained both basic and sulfur containing amino acids and they all produce

positive silver staining reactions [ 65 ]

.

The importance of the basic amino acids has been further substantiated by

evaluations of the relationship between the amino acid mole percentages of

proteins and their ability to stain with silver. The best correlations were achieved when comparisons were made between the slopes of denatured

proteins staining curves and the proteins mole percentages of basic amino

acids, particularly with histidine and lysine [653. A similar correlation

has been observed by Dion and Pomenti C741. Dion and F'omenti suggested that

this correlation may be due to an interaction between lysine and

glutsraldehyde, which was used in their stain protocol. The bound

g 1 u t a r aldehyde could supply aldehyde groups to facilitate the reduction of

51
i o r: i c silver. While this m e c h a n i s m may play a role in the stain protocol

employed by Dion and F'omenti, it is are unlikely to be a -factor in the

Merril and Pratt protocol C653, since that protocol did not employ

glutaraidehyde. Dion and Pomenti also suggested that alkaline conditions

may be important For the Formation oF silver complexes with lysine and

histidine. However the Merril and Pratt protocol utilized acidic

conditions. No signiFicant correlations have been Found between protein

amino acid mole percentages and their ability to stain with silver in a native, or un denatured state C 6 5 3 . This lack oF a signiFicant correlation

with un denatured proteins is probably due to the inaccessibility oF many oF the potentially active amino acid side chains in the undenatured protein

structures.

The observation oF a signiFicant correlation between silver staining

intensity and the mole percent oF lysine is most likely due to the reactive

"amino group" at the terminus oF lysine's side chain. The amino group has

been known to have signiFicant metal binding ability due to its strong

electron-donor qualities and the ligand-Field eFFect oF its nitrogen atoms

[713. However, amino groups involved in peptide bonding and N-terminal

amino groups are in themselves insuFFicient For visualization with silver

stain. IF they were capable oF independently reducing silver ions, all

peptides, proteins, and amino acids would stain positively. Amino groups

involved in peptide bonding and N-terminal atoms may oF some importance For

the intensity oF the stain, as these atoms have been observed to Form 13

diFFerent complexes with copper between pH 1.5 to pH 11.0 C 7 5 3 . Copper may

be reduced under the conditions oF some stain protocols and then be

displaced by silver. Alternatively, silver may also interact directly, but

weakly, with these groups.

The contribution oF histidine to silver staining is not surprising, since

the imidazole groups in the histidine side-chains are oFten important For

metal -b i ndi ng in the metal 1 opr otei ns . The eF F ect i veness oF histidine in

metal binding is probably due to the Fact that imidazole groups are good

electron donors [713. The enthalpy changes in the Formation oF metal- nitrogen (imidazole) bonds are only slightly less than those Found

with metal -nitrogen (amino) bonds [763. The slightly lowered ability oF the

52
imidazole group, relative to the a it: i n o group, to donate electrons -for the

formation of metal complexes may be balanced by imidazole's lower pK. The

lower imidazole group's pK, in contrast to the higher pK of an amino group,

reduces the metal ion's competition with protons for the imidazole's

nitrogen atom's electron -pair [711.

The guanidine group in arginine's side chain has proven to be less active

than either the amino or the imidazole groups in the side chains of lysine

and histidine respectively. Arginine's correlation coefficient was not

found to be significant in studies of comparing staining densities to mole

percent of arginine. This lack of activity of the guanidine group may have

been, in part, responsible for the negative staining reaction of the

peptide angiotensin II which contains the two basic amino acids arginine

and histidine (one residue of each). However, neurotensin, which contains

two arginine residues and one lysine residue, stained well. Cooperative

metal binding effects between active groups may play a role in the staining

process. In angiotensin II the arginine residue is separated from the

histidine by three residues, while in neurotensin the two arginines are adjacent to each other and only one residue separates them from a lysine

residue.

Of the nonpolar and uncharged polar amino acids, only the sulfur containing

amino acids, methionine, cysteine and cystine, showed any silver staining

reactivity with the Merril; Pratt Protocol [65]. Cysteine and cystine were

the only amino acids to stain as an individual amino acids and they may

account for the silver staining properties of the peptide oxytocin.

Oxytocin contains no basic amino acids and its only sulfur containing amino

acids are two cysteine residues. The ability of cy stein y 1 side-chains to

form complexes with silver ions is well known. At the low pHs utilized in

this protocol, the predominant species is Ag (HCys) z*", C71 3. It has been

suggested that the ability of reducing agents [including: thiosulfates,

sulfides, torch ydrides, cyanoborohydrides, mercaptoethanol, thiogly colic

acid, cysteine, tributylphosphine reducing metal salts (such as FeCl 2 ,3nCl 2 and T i C

1

3 ) and dithiothritol] to intensify silver stains may be

related to the generation of thiol groups in cysteine residues [773.

However, proteins that contain no cysteine or proteins with an alkylated

53
c y s t e i n e (

s

) were also affected by these reducing agents in some stain

protocols [721.

Methionine's ability to participate in the silver staining process has been

demonstrated by silver staining of methionine homo polymers. Methionine may

also be responsible for the staining of the peptide qastrin. Gastrin

contains no basic amino acids and only one methionine residue. In general

the thioether sulfur atoms in the methionine residues are weaker electron

donors than the sulfhydryl sulfur atoms in the cystiene residues. The only

metal ions that have been observed to bind to the thioether's sulfur atoms

are those with electrons in the d s and d 10 configurations (Pd -**, Pt*-, Ag-,

Cu.% and Mg*""). The affinity of sulfur ligands for metal ions may be

explained by the higly polarized state sulfur atoms achieve during

interactions with small metal ions containing high charge densities.

Sulfur's electron distributions and energies enhance the enthalpies of

metal ion bonding (they have high crystal field stabilization energies).

There may also be electron resonance bonding in the metal-sulfur bond [711.

Insignificant staining correlations were observed between staining

densities and mole percentages of the sulfur containing amino acids

methionine and cysteine [653 . This observation may indicate a relatively

minor silver staining role in proteins containing large numbers of basic

amino acids. However, this poor staining correlation is somewhat of a paradox since poly-methionine stained with a higher silver density than the

basic amino acid homopolymers [653. This paradox may be explained by a strong requirment for cooperative effects between sulfur atoms and silver

atoms which may be disrupted in heteropolymers.

5 Properties Of Silver Stains

5.1 Color Effects

Most proteins stain with monochromatic brown or black colors. However, the

ability of silver to produce colors was first noted when certain

lipoproteins tended to stain blue while some glycoproteins appeared yellow,

brown or red in a study of cerebrospinal fluid proteins [783. This color

effect is most likely an analogue of a photographic phenomena first

54
described by Herschei in 1840 [53,7?]. Herschel noted in 1840, that if he

projected the spectrum of visible light obtained by passing sunlight

through a prism onto a silver chloride-impregnated paper, the colors o-f the

spectrum appeared on the paper, particularly a "full and fiery red" at the

focal point of the red light [791. Since these observations by Herschel it

has been found that the color produced depends on three variables: the sice

of the silver particles, the refractive index of the photographic emusion

or electrophoretic gel, and the distribution of the silver particles. In general, studies with photographic emulsions have shown that smaller grains

(less than 0.2 microns in diameter) transmit reddish or yellow-red light,

while grains above 0.3 microns give bluish colors, and 1 ar ger grains

produce black images [681. Modifications of the silver staining procedures,

such as lowering the concentration of reducing agent in the image

development solution, prolonging the development time, adding alkali, or elevating the temperature during staining often enhance color formation.

Some silver stain protocols have been developed to produce colors that may

aid in identification of certain proteins [73,80-813. Production of color

with silver stains depends on many variables. Amino acid side groups play a major role in color formation [733. Furthermore, variations in protein

concentration and conditions of image development may also produce color

shifts. Color-enhanced silver stains tend to become saturated at low

protein levels and often produce negatively stained bands or spots. These

factors tend to make quantitative analysis with silver stain protocols that

enhance color effects more difficult.

5.2 Protein Specific Silver Stains

Silver stains can demonstrate considerable specificity. Hubbell et a 1

.

stained nucleolar proteins with a histological stain C 4 6 3 , while Gambetti

et a 1 . adapted a silver stain specific for neurofilament polypeptides [543.

Many silver stain protocols detect not only proteins but also DNA, [82-843,

lipopoly saccharides [853, and polysaccharides [863. In a study of

erythrocyte membrane proteins, sialogly coproteins and lipids stained yellow

with a silver stain protocol, while other membrane proteins counterstained

with Coomassie Blue [873. All silver-stains do not detect proteins such as calmodulin or troponin C. However, pretreatment with gluteraldehyde often

55
permits positive silver staining of these proteins [38], Histones may also

tail to stain with silver. Fixation with -formaldehyde coupled with

simultaneous prestaining with Coomassie Blue partly alleviates this

problem. However, even with this -fixation procedure sensitive for histones

is decreased 10 -fold compared with detection of neutral proteins C891.

Another example of differential sensitivity was demonstrated in a study

utilizing four different silver stain protocols to stain salivary proteins.

Different protein bands were visualized with each of the stains [901.

5.3 Quenching Of Autoradiography by Silver Stains

Quenching of 14 C-la belled proteins is minimal with most of non-diamine

silver stains and even the most intense diamine stained, 14 C-labelled

proteins can be detected by autoradiography with only a 507. decrease in

image density. This loss of autoradiographic sensitivity can generally be

compensated for by longer film exposures. However, detection of 3 H-labelled

proteins is severely quenched by all silver stains. Destaining of the

silver stained gel with photographic reducing agents can often permit

detection of as much as half of the fluorographic density of 3 H-labelled

proteins, providing that the initial staining was performed with a non-diamine silver stain. Many diamine stains continue to quench, even after treatment with photographic reducing agents, so that fluorographic

detection of 3 H-labelled proteins is not feasable. This impediment to 3H

detection with diamine stains is likely to be due to a greater amount of

residual silver deposited in the gels by the diamine stains, which block

the weak -beta emissions from 3 H . Residual silver has been demonstrated in

gels that have been cleared by photographic reducing agents by the faint

silver image of the protein can be observed after drying the gel with heat.

Silver has also been demonstrated in these "cleared" gels by electron beam

analysis [913.

5.4 Sensitivity

Silver stains offer the most sensitive non-radioactive method for detecting

proteins separated by gel electrophoresis. They are 100-fold more sensitive

than the Coomassie stains for most proteins [18-193. Chemical-development

56
silver stains are in general, more sensitive than photo-development silver

stains. This loss in senstivity may be compensated for by the ability of

photo-development stain to produce an image within 10 to 15 minutes after

gel electrophoresis C 6 4 3 . Attaining high sensitivities with silver stains

requires care in selecting reagents, small traces of contaminants may cause

a loss of sensitivity and result in staining artifacts. Artifactual bands * with molecular weights ranging from 50 to 68 kiloDaltons have commonly been

observed in silver stained gels. Evidence has been presented indicating

that these contaminating bands are due to keratin skin proteins [923. Water

used to make solutions should have a conductivity of less than 1 mho.

6 Quantitation With Protein Stains

6.1 Quantitation Using Organic Stains

Face k a s de St. Groth et al . demonstrated an accuracy of plus or minus 1

0

7.

in measuring the concentration of a protein in the range of 0.5 - 20 ug/cm

using Coomassie Blue P.250 C 1 2 3 . He noted that, although individual proteins

displayed linear relationships between absorbance and concentration, the

slopes differ for each protein. This variation in Coomassie Blue staining

now appears to be related to the mole percent of the basic amino acids in

the protein [301. Therefore, a standard curve must be produced for each

protein assayed and quantitative comparisons limited to equivalent protein

spots on two-dimensional or equivalent protein bands on one-dimensional

electrophcretograms. This is especially true for metachromatically staining

proteins, which often display an additional complication, in that their

bands fade more rapidly than the non-metachr omat i cal 1 y staining proteins.

In one study with Amido Black, certain metachromati c histone bands faded by

30-507., while other protein bands in the same gel faded by only 5-157. [341.

This fading reflected a disproportionate loss of stain, rather than a loss

of histone protein, since the bands could be restained. In the case of

smaller proteins such as insulin, decreases in band densities have been

demonstrated to be due to the loss of protein during staining [123. Another

problem in utilizing Post-electrophoretic organic stains occurs because

diffusion of the dye into the gel is slow. This results in "ring-dyeing"

if staining is terminated prematurely [153. In "ring-dyeing", the stain

57
concentration is less in the center of a band or spot than at the edge, due

to the insufficient diffusion of dye molecules. Regional nonstoichiometric

processes, as occur in "ring-dyeing", require careful control of staining

parameters to assure that they are not present in protein bands or spots

that are to be quantitatively analyzed.

6.2 Quantitation Using Silver Stains

A reproducible relationship between silver stain density and protein

concentration has been found with most silver stain protocols. The linear

portion of this relationship extended over a 40 -fold range in

concentration, beginning at 0.02 nanograms per mm 2 for most proteins

[ 19,57-53,632. Protein concentrations greater than 2 ng/mm 2 generally cause saturation of silver images, resulting in non-linearity above that

concentration. Saturation can usually be recognized by bands or spot with

centers which are less intensely stained than the regions near the edges.

This effect is similar to the "ring-dyeing" noted with some of the organic

stains. An often quoted report by Poehling and Neuhoff [931 states that

"Silver does not stoi chi ometr i cal 1 y stain proteins, unlike Coomassie Blue".

However, their own silver-stain data actually is linear over a 30-fold

range in protein concentration, while their Coomassie Blue data is only

linearity over a 20-fold range [20,57].

Employment of Curve-fitting techniques, as described by Coakley and James

[941 for the analysis of curvilinear relationships found in the Folin-Lowry

method of protein estimation [953 may be utilized for quantitative analysis

of silver stain curves. With careful measurement of total stain densities,

estimates of relative protein concentrations have been made over a 220 fold

concentration range with six purified proteins [653.

Plots of silver stain densities versus protein concentrations produce

different staining curves for each proteins studied [18,57-58,653. Protein

specific staining curves have also been observed with the organic stains,

including Coomassie Blue [3 0,123, as noted above, and with most protein

assays such as the commonly used Lowry protein assay [953. The observation

that each protein produces a unique density verses concentration curve in

58
these studies, illustates a dependence on specific reactive groups

contained in each protein. Furthermore, the occur a nee of protein-specific

curves argues against a stain mechanism that depends on some fundamental

subunit common to all proteins, for example the peptide bond, or a unique

element in each protein, such as the terminal amino acid. A stain that

depended on a subunit, such as the peptide bond, would result in similar

staining curves for all proteins, when the density of staining for each of

the protein bands or spots was plotted against the mass of protein

contained in each of the bands or spots. Similarily, a stain that was based

on a reaction with a unique element in each protein, for example the

terminal amino group, would produce similar plots for each protein when the

stains densities were plotted against the number of molecules contained in

each band or spot. It is possible that these protein -specific curves may be

utilised to differentiate proteins and to provide insights concerning the

reactive groups responsible for the staining reactions. The importance of

the basic amino acids, particularly lysine and histidine as discussed in

the section " Staining of Peptides and Proteins with Known Sequences "

illustrates the use of these proteins specific staining curves. It indicates the need for a careful choice of a "standard protein(s)" if this

stain is used quantitatively to estimate protein consentrations. A protein

containing an abnormal large number of stain reactive groups would produce

a curve which would tend to underestimate the concentration of proteins

containing normal numbers of reactive groups. A similar correlation between

the intensity of Commassie Blue staining and the number of basic amino

acids in proteins [29,30] caused Tal et al. to suggest the use of egg white

lysozyme rather than the more commonly used bovine serum albumin as a protein standard. This suggestion is based on their observation that the

basic amino acid content of proteins ranges between 10-17 mole percent,

with a modal content of 13 mole percent C 3

0

3 . Egg white lysozyme has a basic amino acid mole percent of 13.2 while bovine serum albumin has a basic amino acid content of 16.5 mole percent. For similar reasons, egg

white lysozyme may also prove to be an optimal standard for quantitative

silver stain applications.

6.3 Quantitative Inter-gel Protein Comparisons

59
The seen ranee of Protein specific staining curves with most staining

protocols requires that quantitative inter-gel comparative studies limit

comparisons to homologous protein bands or spots on each gel. For example,

the actin spot on one gel can be compared with an actin spot on another

gel, but not with a transferrin spot. Quantitative inter-gel comparisons

requires the presence of reference proteins for the normalization of spot

or band staining densities. One scheme for normalization utilizes

"operationally constitutive proteins", a subset of proteins contained in

each gel that have constant intra-gel density ratios to each other in all

of the gels in a study. The sum of the densities of the "operationally

constitutive proteins" in an arbitrarily designated "standard gel" are compared with the sums of the densities of the constitutive proteins in all

other gels, and a specific normalization factor is determined for each gei.

These gel specific normalization factors are then utilized to correct the

densities of all the proteins on each of the gels to those of the standard

gel. This scheme corrects for variations in staining, in image

digitization, and initial protein loading; a variation of initial protein

loading of up to 1

0

- f o

1

d may be tolerated C

5

7

,

6 3 ]

.

7 Conclusions

The introduction and development of electrophoretic techniques has produced

ever more powerful means of resolving proteins from complex mixtures.

Development of these separation techniques has been paralleled by the

devel op men t of protein detection methods with ever-increasing sensitivities.

Techniques have progressed from direct observation of protein-coated

micro spheres and colored proteins, to the detection of proteins by their

absorption of ultraviolet light, the observation of schlieren patterns,

and, more recently, by direct staining with organic, fluorescent and silver

stains.

During this technical evolution, sensitivity has increased from the

milligram to the tenth of a nanogram level. This range of sensitivity

coupled with high resolution separation techniques, now permits clinical

studies of proteins in body fluids that were not possible even a decade

ago. Similar sensitivities can be achieved with proteins labeled to high

60
specific activities with radioactive tracers, however, raadioactive

labelling is often not possible in animal and human studies for economic

and, in the latter case, ethical considerations.

Many of the protein staining techniques may be employed quantitatively,

provided that their methodological limitations are respected. Most protein

stains and autoradiographic methods exhibit protein-specific quantitative

responses. Optical density /concentration relationships are usually linear

over a thirty- to forty -fold range in concentration and quantitation may be

extended beyond this range by the use of curve fitting techniques.

Protein -specific staining slopes are indicative of a dependence of these

staining methods on the content of specific groups within each protein.

These protein -specific staining slopes may be utilized to differentiate

proteins, and emphasize the need to limit quantitative comparisons to

homologous proteins. So long as intergel studies are confined to

comparisons of homologous proteins and observations are made within the

linear range of the detection procedures, valid quantitative results may be

r. rjf si npH t

7 h i s !! o r k mas supported by the Intradural Program of the HIMH and the

D o

n

a 1 d a nd Charity Yarborough Fund

.

5 References

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[5] Picton, H. and Linder S.E., Jour . Chen . So c

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65
Susan C. Olson, and Peptide Separations:

Carl R. Merril

Section on Biochemical Genetics Techniques Reviewed

Clinical Neurogenetics Branch

National Institute of Mental Health

Bethesda, Maryland 20892

Numerous techniques, both electrophoretic and

chromatographic have been developed to separate peptides

from complex mixtures. However, detection of low

concentrations of small molecular weight peptides in

biological material, as well as separation of

proteolytic digests of larger proteins continues to

offer difficulties. Various chromatographic techniques

such as paper chromatography, paper electrophoresis,

thin layer chromatography, thin layer electrophoresis,

polyacrylamide gel electrophoresis and high performance

liquid chromatography have been employed for the

analysis of peptides. But each of these techniques has

limitations in terms of the range of peptide sizes that

can be successfully separate. Many of the methods also

lack the ability to adequately separate structurally

related molecular species, such as intermediates in the

biosynthetic pathway and posttransl ati onal 1 y modified

pepti desC 1 ]

.

i Paper Chromatography and Electrophoresis

Since the 1944 introduction of paper chromatography by Consden, Gordon,

and Martin, it has proven to be a simple chromatographic technique. It readily provides qualitative information and depending on the method of

detection it is possible to obtain quantitative data.

Samples to be analyzed are usually dissolved in organic solvents with

low boiling points such as acetone, ethanol, or chloroform C 3 3 . Ionic

and polar molecules separate fairly well in this system, however its

ability to separate nonpolar molecules is limited C 4 3 . Solvent systems

containing butyl alcohol and acetic acid or systems containing amyl

alcohol are used for separation of basic peptides, whereas pyridine is

a good solvent for the separation of acid peptides C41. For highly

efficient separation of peptides on paper it is important for the

analyzed substances to be free of larger proteins, free amino acids and

inorganic ions [ 4 ] „ These interfering substances may cause poor

66
resolution, spot distortions, and aberrations in reactions used for

peptide detections C5]. Humidity in the chromatographic chamber and

often the complexity of the peptide mixture may also affect the

reproducibilty of the Rf values for peptides in individual solvent

systems C43.

Many types of papers are used for peptide analysis: standard cellulose,

papers containing carboxyl groups, ion-exchange resins, cellulose

ion-exchange, alumina or silica gel adsorbents and glass fiber papers

C 3 3 . There are four methods of applying solvents based on the

direction of solvent movement; ascending, descending, horizontal, and

radial techniques C33. These techniques may use any one of three

different types of solvent systems for their development: an aqueous

stationary phase; a stationary hydrophilic organic solvent ; or a stationary hydrophobic organic solvent C 3]

.

Ultraviolet light is primariy used for the detection of peptides on paper C33. In addition, peptides may be detected by: enzymatic

detection C33; ninhydrin staining [5,63; production of a fluorescent

product with fluorescamine [73 or o-phthaldehyde [53; reaction with the

Ehrlich reagent which detects tryptophan and tyrosine residues; and

radioactivity using a labelled modifier [83. Dintrophenyl [DNP3 amino

acids can not be sucessfully separated by paper chromatography since

they have a tendency for trailing, and also their migration on paper

depends on the quantity of amino acids applied and on the presence of

other DNP amino acids [93.

High voltage paper electrophoresis is a rapid and reproducible method

for the separation of peptides. Not only is this procedure faster than

paper chromatography, the samples need not be desalted unless very

large samples are used, or unless the salt concentration is much higher

than physiological [103. However, this procedure requires special

attention to such details as pH, temperature, voltage, and type and

length of paper. This method uses the same types of papers as those

described for paper chromatography, also the detection methods of

67
peptides are similar. As little as 1-2 nmoles of peptides may be

detected by f 1 uor escami ne staining after high voltage electrophoresis

on paper [111.

Although, this procedure is highly useful for the resolution of small

peptides, it does have several drawbacks such as: heat dissipation and

evaporation of buffer from the paper electrophoretagram CIO]; the

inability to examine peptides that have low solubility in the

electrophoretic bufer; and the trailing of intermediate and larger size

peptides on the paper C113.

Many researchers used a two dimensional system of paper chromatography

fallowed by high voltage paper electrophoresis to analyze tryptic

digests of various proteins [12—153. This system is very reproducible.

Katz C143 found that ascending chromatography with n-butanol /aceti

c

acid /water [4:1:53 followed by electrophoresis with a pyridine acetate

buffer at pH 3.7, allowed one to detect the replacement of a single

amino acid. This chromatography system separated the polar from the

nonpolar peptides, and the electrophoresis separated the polar peptides

according to charge.

A number of researchers feel that the most reproducible method of

peptide separation involves electrophoresis on paper followed by paper

chromatography [16,173. Ingram [173 using paper electrophoresis at pH

6.5, followed by ascending chromatography in n-butanol/acetic

acid/water [3:1:13 found this technique also allowed for the detection

of a single amino acid difference between normal and sickle cell

hemoglobins of man.

2 Thin Layer Chromatography and electrophoresis

Although the principle of thin layer chromatography [TLC3 was described

by Izmailor and Schraiber in 1938 [173, it was not until 1956 that it was introduced as a technique for analytical adsorption chromatography

[183. Thin layer chromatography is a relatively inexpensive, fast, and

simple method which can separate complex mixtures on a single thin

68
layer plate. This method can be used -for the determination of

appro:: i mate molecular weights of peptides and small molecular weight

proteins. In addition, this procedure can be used to determine the

purity of peptides as well as for the analysis of peptides when only

minute amounts are available C191. Also the starting material, the

intermediates, and the end products in peptide synthesis can be

examined rapidly by TLC [20].

Once TLC plates have been prepared , the technique of chromatography is

similar to that used for paper chromatography. Commercially prepared

plates are available, they offer the advantage of highly uniform layers

and good r epr oduc i b i 1 ty . The following types of prepared plates are available: silica gel Kieselguhr, alumina [acid, basic, or neutral],

magnesium silicate, calcium sulphate, zirconium phosphate, and

hydroxylapaptite [21]. Organic materials used on the plates include:

various derived cellulose such as carboxymethyl , d i ethyl ami noethyl

,

phosphate and various impregnated cellulose such as pol yethy 1 eni mi ne impregnated; Sephadex, free and substituted; Avirin and Avicel which

are microcrystalline celluloses; polyamides such as nylon and perlon;

and powdered ion-exchange resins [21].

Thin layer chromatography depends on differences in charge, molecular

weight, hydrophobicity, and adsorption behaviors for the separation of

peptides [22]. The presence of foreign substances as well as sample

overloading can cause poor resolution, spot distortions, and aberration

in colored reactions used for peptide detections [5]

.

Like paper chromatography, TLC permits rapid qualitative results, but

exact quantitative determinations are difficult to obtain. Peptides

can be located on TLC plates by UV spectroscopy [5], ninhydrin staining

[23]

, fluorometry [23], radioautography [5] and di n i tropheny 1 at i on

[24]

. Location of these peptides with ninhydrin, f 1 uor escami ne or o-phthaldialdehde permitted the detection of picomole quantities of

peptides [23]. It is important to note that only peptides detected

using fluorescamine can be recovered from their layers without

destruction of any amino acids thereby allowing the peptide to be

69
subjected to further analysis [23,24,251. Detection of peptides with

n inhydrin is limited because the molar responses decrease with

increasing chain length, and because N-acylated peptids are usually

non re active. Also it fails with cyclic peptides unless they contain

free amino groups in their side chains C81.

Peptides can be isolated from thin layer plates either by solvent

extraction and filtration or by electrophoretic elution of the peptides

C261. After elution [221 the fractions can be quantitatively analyzed

by ultraviolet spectrometry [271, by fluorimetry [281, by calorimetry

after chemical reaction [291 or by weighing [303

.

Thin-layer chromatography has been used for the studies of small

peptides [301 as well as for peptide mapping [32,33,34,353. Burzynski

[323 found that tritiated dansly chloride labelled peptides analyzed on polyamide plates followed by autoradiography and densitometry allowed

detection of 2.5 :< 10-14 moles of peptides.

Reverse phase TLC plates made with silanized silica gel impregnated

with detergents are also available for the analysis of peptides [333.

The type and concentration of the detergent determines the separations

that can be achieved. The interaction of the hydrophobic side chain

and functional groups of the detergent with the amino acids controls

the migration of the peptide on the plate. Peptides with hydrophilic

or basic amino acids run with the solvent front while peptides with one

or more hydrophobic amino acids are retained [363. This reverse phase

TLC system [using Whatman KC18F plates3 provided a rapid check of HPLC

separation, and detection of the material with ninhydrin verified that

the optical density peaks were actually peptides [373. Before polar

samples can be analyzed in this reverse-phase TLC system, an ion

pairing reagent must be added to the mobile phase. This reduces the

migration of the polar solutes and minimizes spreading of the spots

[373. Peptide containing hydrophobic and basic residues have lower Rf

values because of their increased interaction with the stationary

phase. Therefore, an increase in the amount of acetonitrile or methanol in the mobile phase results in a higher Rf value. As observed

70
for reverse phase high perforamnce liquid chromatography, acetonitrile

was -found to give more efficient separation of peptides in reversed TLC

than was achieved with methanol [37]. It is possible to use TLC data

to develop a more effecient HPLC system, however TLC could still be the

desired method when a very large number of samples are to be analyzed.

Two dimensional thin layer methods in which peptides are separated by

electrophoresis horizontally followed by chromatography [ascending] is

commonly called thin-layer electrophoresis [39], This procedure has

been used widely for peptide map analysis [38,39,40,41]. Thin layer

electrophoresis requires only a simple apparatus which provides a cheap

and satisfactory alternative to more expensive and sophisticated high

performance liquid chromatography machinery. Also, this method does

not require the use of high voltage equipment which are capable of

dissipating large amounts of heat [42].

3 Polyacrylamide Gel electrophoresis

In 1967, Shapiro and CQWorkers [43] demonstrated that the migration of

denatured polypeptides on polyacrylamide gels correlated closely with

their length. However, application of this method for determination of

the molecular weight of peptides less than 10,000 on sodium dodecyl

sulfate polyacrylamide gel electrophoresis [SDS-PAGE] has encountered

many problems. The importance of the intrinsic charge of the peptide

on the influence of SDS binding was observed by Swank and Munkries

[44], Assuming that all peptides bind 1.4 mg SDS per mg, the smaller

peptides bind relatively less SDS on a molar ratio; therefore the

intrinsic charge becomes more important at these lower molecular

weights [44]. Also, factors such as shape, final net charge and side

chain modifications [44,45] also greatly influence the migratory

behavior of peptides on polyacrylamide gels. Many approaches have

been used to obtain better resolution of low molecular polypeptides in

SDS-PAGE , they include: addition of urea to the gels [44,45,46,47];

increasing the concentration of acrylamide and/or crosslinkers

[44,47,481; altering the buffer composition [44,47]; altering the pH

[11,49]; using linear acrylamide gradients [47,50]; and addition of

71
sucrose [47].

Swank and Munkries were among the first to investigate conditions for

the maximal separation of peptides [443. First, they added urea to the

standard SDS system [433 | urea increases the solubility of some peptides, and it also reduces the effective pore size in acrylamide

gels. In addition, urea may decrease the size of the detergent

micelles. This may help to overcome the problem of coelectrophoresis

of small peptides, since peptides less than a certain length all end up

in complexes of peptides and detergent of the same size and charge

[513. And finally the presence of urea allows one to use higher

levels of cross-linker which improves resolution of oligopeptides [443.

The Swank and Munkrie system makes use of higher acrylamide

concentrations which improved the separation of peptides [443. Another

advantage came from the replacement of sodium with Tris, a cation with

much lower mobility thus less heat is produced in the system. Tris may

also contribute a small amount of buffering capacity to the system at

pH 6.? [443.

The major drawback of this system is the long running time which

results in diffuse protein bands, thus decreasing resolution. The

broadening of protein bands also results from the use of a uniform

separation gel without a stacking gel and a continuous buffer system.

And finally this system does not allow for accurate moleclular weight

determinations between 10,000 and 20,000.

Although Swank and Munkries [443 developed a procedure for separating

peptides between 1000 and 10,000 daltons on tube gels, this method was not applicable to slab gels [453. This led Burr and Burr [453 to

modify this technique with the addition of stacking gel to allow

separation of peptides between 1500 and 10,000 daltons using slab-cast

gels. Linearity beteen mobility on the gel and molecular weight of the

proteins was observed to 25,000 daltons.

More recently, a system which depended on buffer composition and

72
concentration and which was not critically dependent on acrylamide

concentrations gave accurate molecular weight determinations -for low

molecular weight proteins from approximately 2500 to at least 90,000

[521. The separating gel contained 5 to 187. acrylamide, all with 57. crosslinker concentrations. Reproducible results required 8M urea in

the separation gel, omitting urea or lowering the concentration of urea

gave inaccurate molecular weight determinations tor proteins between

10,000 and 20,000. However, the improved resolution of this system is

primarily due to the buffer system used. The buffer of the stacking

gel contains a relatively low-mobility acetate ion, and the buffer of

the separating gel contains the high mobility strong anions, sulfate

and chloride, as the leading and trailing ions respectfully. The

proteins will remained stacked as long as their mobilities are between

those of the leading and trailing ions. The organization of the ions,

acetate, sulfate, and chloride, at the beginning of el ectrohor esi

s

determines the unstacking of the lower molecular weight proteins. The

sulfate/acetate/protein complex is stacked but as electrophoresis

precedes the chloride ion overtakes the complex, the acetate and

proteins then become successively unstacked [523. This discontinuous

buffer system also allows one to load dilute samples on the gel since

stacking results in concentration of the sample. Furthermore,

electrophoresis is complete within 4-5 hours thus limiting the amount

of diffusion of the polypeptides within the gel.

Kyte and Rodriguez C 5 3 3 also developed a method for separating peptides

that contains a stacking gel; this system controls the stacking of the

proteins by altering the pH. In this system the acrylamide

concentration was not found to be critical, the length of a peptide was approximately the same when determined using 12.5 and 207. polyacrylamide gels. However the Ferguson plot analysis indicated that

the gel with the higher percent acrylamide gave the more accurate

peptide length. These investigators determined that higher

concentrations of protein added to the gels caused the occurence of

electrophoretic artifacts and diffuse protein bands [533. The major

drawback of this system is that it only provides high resolutin for

peptides between 25 and 250 residues in length and a large majority of

73
the peptides to be studied contain less than 25 residues.

More recently a SDS-PAGE system using a 10 to 187. linear gradient

containing 7M urea with an acr y 1 ami de : b i sacr y 1 ami de ration of 20:1, a 0

to 107. sucrose gradient, and Laeml 1 i discontinous buffer [54] was described [473. Proteins ranging in molecular weights from 1500 to

25,000 were highly resolved. Although the electrophoresis running time

is long [15 h] the high crosslinking ratio lowers the diffusion of the

peptides in the gel as well as limits the leaking of the polypeptide

from the gels during staining [473.

Two polyacrylamide gel electrphoretic systems which do not contain

detergent were described for the separation of pept i des . [ 49 ] . The

acidic system contains 507. acrylamide, has a pH of 3.0 and containes

urea in the stacking gel but not in the resolving gel. Peptides with

one positive charge to about 180 daltons were resolved. To separate

peptides which have a net negative charge at pH 3.0 or are insoluble at

this acidic pH, an alkaline peptide gel was used [493. This gel has a pH of ? and also has urea in the separating gel. This system has been

used to study histone proteins [553.

As early as 1973 iodinated and uniodinated Angiotensins were separated

on gel electrophoresis on the basis of charge [563. Recently a polyacrylamide gel electrophoresis system which separated Angiotensin

I, II, and III based entirely on net charge rather than size was demonstrated [503. The system was run a pH 6.41, positive polarity,

used gel concentrations ranging from 24 to 407. acrylamide, 27. bisacrylamide and used a discontinuous buffer system.

Two dimensional electrohoresis has also been used to separate peptides

1573. One must take precautions to prevent protien loss from gels

during equilibration and loading steps. If the isoelectric focusing

strips are equilibrated longer than one minute prior to loading on SDS

slabs there was a significant diffusion and loss of the small molecular

weight proteins from the gel strips. The use of a higher percent

acrylamide in the first dimension effectively decreased diffusion of

74
the peptides [573. Hashimoto et.al. [473 showed that a higher

crosslinking ratio [20:1, acr y 1 ami de : b i sacry 1 ami de3 also lowered the

leakage of the small peptides from the gels. Blattler and coworkers

[483 investigated the acrylamide and bisacrylamide concentration

effects on the properties of the gels. Altering bisacrylamide

* concentration affects maximum pore size of the gel while changing

acrylamide concentrations affects the average pore size [483. They

noted that too much bisacrylamide produces weak, paste-like gels that

are rigid as well as brittle. Prelim in ary evidence from Blattler's lab

also indicates that catalyst conditions are important for producing

uniform quality gels [483.

The small molecular weight peptides which occur in very low

concentrations in body fluids that are separated by SDS-PAGE can be

analyzed by silver stain [583. However, rapid diffusion and loss

during the staining and destaining procedures may limit quantitative

studies of the low molecular weight peptides [113. But, these peptides

may be retained in the gels by fixing them chemically as opposed to

methanol-acid denaturation [593. For example, glutaraldehyde which

reacts with primary amino groups and guanidium groups can be used to

fix peptides in the gels [573. It should be noted that peptides cannot

be fixed by glutaraldehyde on isoelectric focusing gels and

subsequently silver stained because carrier ampholytes are fixed in

the presence of glutaraldehyde and will also stain [603. Formaldehyde

[613 can also be used to fix peptides in the gels, but proteins which

do not have a free amino group are lost from the gels. [493.

Although the silver staining procedure is much faster than staining

with Coomassie Blue, Coomassie Brilliant Blue R-250 is often used to

stain peptides between 1500 and 10,000 [453. Addition of copper

sulfate [0,57.3 aided in the retention of the peptides in the gel [453 .

In addition, since peptides are lost from the gel during acid

precipitation, fixation of the peptide with 1.257. glutaraldehyde

permitted their use of Coomassie blue procedure to detect peptides

with molecular weights as low as 1000 [503.

75
Other methods of detecting peptides include: autoradiography preceeded

by glutaraldehyde fixation [491; spectrophotometric detection of the

tyrosine or tryptophan residues [621; separation of peptides by

isoelectric focusing on polyacrylamide gels followed by fixation on glutaraldehye impregnated paper and identified using immunoperoxidase

staining procedures C 6 3 3 ; and fluorescent staining C 6 4 3 . However, there

were problems using f luorescamine. Modifications of primary amines of

peptides with f 1 ucrescami ne increases the net negative charge of the

peptide resulting in excessive rapid migration , therefore higher

acrylamide concentrations C 12—16X3 were necessary to separate peptides

from the fluorescent front [641. Also pH affects the mobility and

resolution of peptides modified with f 1 uor escami ne [11], only at the

intermediate pH [8.9 to 9.4] are the peptides well resolved without

trailing. At lower pH the peptides move too slowly to achieve

significant separation whereas at higher pH values they move too fast,

producing trailing.

4 High Performance Liquid Chromatography

High performance liquid chromatography [HPLC] has become a very

important analytical tool for isolation and separation of peptides for

biological, chemical, and biophysical studies. Reliable HPLC systems

were developed based on peptide characterenstics such as length of the

peptide chain, hydrophobicity , and nature of the individual amino acids

[65]. The column supports have a definite pore structure which depends

on the type of liquid chromatography [ion exchange, size exclusion, or partition /adsorption] being considered [66],

High Performance Liquid Chromatography offers distinct advantages over classical chromatography such as increased resolution, speed,

reproducibility, and high recoveries of limited quantities of samples.

High sensitivity may also be achieved when the peptides are detected by

radioimmunassay, fluorometrically or spectrophotometrically. Since

amides absorb in the 210-230 nm region of the spectrum, polypeptides

may be detected spectr ophotometr i cal 1 y thereby achieving high

sensitivity (50 to 100 nanograms). Another advantage of HPLC is that

76
it is nondestructive thereby allowing recovery of biological materials

or recovery of material desired for sequencing.

4.1 High Performance Ion Exchange Chromatography

High Performance Ion Exchange Chromatography C HP I EC 3 currently plays an • important role in the separation and purification of peptides: cationic

exchangers are used for neutral and basic peptides and anion exchangers

are used for neutral and acidic peptides. The columns of HPIEC have

two types of support materials, either totally organic materials or surface modified inorganic supports. Peptide separations have been

achieved on either silica- based or pol ystyrene-di vi n 1 ybenz ene based

microparticulate pourous ion-exchange stationary phases C671.

An important factor in ion- exchange chromatography is the support pore

diameter, it greatly influences the separation of peptides on the

column. The pore diameter must be sufficiently large enough to allow

the penetration of large molecules as well as allow for maximun

ion-exchange capacity. Temperature can effect peptide retention and

selectivity as well, therefore optimal temperature will vary for

different peptide mixtures C 6 8 3 . The resolving power of an ion -ex change column is also affected by ionic strenth and pH. Cachia

E 6 9 3 found that a mixture of peptides which vary with respect of net

charge and basic residue composition could be separated on a carboxymethyl cellulose C CM 3 300 weak cation- exchange column. The

number of ionizable groups present on the CM supports were increased

with increasing the pH Ci.e. 4.5 to 6.5], thus increasing the resolving

power, but at the same time more than doubled the time necessary to

complete the separation. Also, an increase in solutes, such as potassium chloride, decreases the retention time of proteins but the

separation between structurally related and similarly charged

peptidesis is maintained.

Elution of a peptide bound to an ion-exchange column can be achieved by

changing the ionic strength or the pH of the mobile phase C 7 0 3

.

Peptides have been successfully separated on HPIEC using mixtures of

77
acetonitrile and tr i ethyl ammoni urn acetate [TEAA3 buffer as the eluent

[711. This buffer system had minimal absorbance in the low UV allowing

detection of the peptides in the 210-225 nm range. Also the TEAA is a volatile buffer, therefore the buffer can be removed during

lyophilization making the system useful for preparative chromatography

and subsequent direct bioassay. Other than UV absorption, peptides

eluting from the columns can be detected by a variety of other methods

including: fluorescence methods [723, differential ref ractometer [733,

radioactivity detectors [733 and ninhydrin based monitoring systems

[74 3 .

One advantage of HPIEC is that column length has minimal influence on resolution [753 therefore protein samples may be eluted in a smaller

volume of mobile phase and detector sensitivity will be greater [703.

Also speed of enzyme recovery on a smaller column is important when

attempting to purify labile proteins [703.

Applications of HPIEC for peptide separations include: separation of

proteolytic digests [76,773; analytical peptide mapping [78,793;

analysis of peptides hormones [80,813 and the detection of abnormal

hemoglobins in the newborn [823.

4.2 High Performance Size Exclusion Chromatography

Many investigators have found that size exclusion chromatography [SEC]

or gel permeation high performance chr omatogaphy [GP-HPC3 yields a linear relationship between void volume [i.e. retention time] and log

molecular weight of a peptide [1,70,833. Furthermore because peptides

of the same molecular weight vary in molecular shape due to functional

groups along the side chains, and hydrodynamic volume, retention in SEC

is also shape dependent. Thus, calibration curves cannot determine the

molecular weight of a peptide of unknown shape with any degree of

certainty. Therefore it is important that the mobile phase of SEC must

be one that will eliminate partitioning and/or absorption on the

packing material.

78
Rivier [83] points out that without addition of a detergent, adjustment

of the pH and the ionic strength o-f the eluent is necessary -for the

separation o-f peptides according to size. A linear relationship was

-found between log molecular weight [1,000 - 44,000] versus retention

time when using a F'ac 1-125 [ Water Associates] in the presence of

triethylammonium phosphate or -formate bu-f-fer [pH < 3 3 in the presence

o-f 15-30 7. acetonitrile. This HPGPC system required a noncompressi b 1 e support with minimal non-speci -f i c adsorption and controlled pore size.

The advantage o-f this system include biological compatibility, low

ionic strength and UV transparency.

Richter and coworkers [843 also developed a procedure tor molecular

weigth determination ot peptides using a Bio-Sil Tsk 20 and Bio-Gel T5K

125 columns. In the presence ot 6M guanidine hydrochloride, standards

trom 1000 to 10,000 were inversely correlated to the logarithms ot

their molecular weight on the TSK 20 columns. Two combined TSK 125

columns gave reliable separation ot peptides in the molecular weight

range trom 800 to 3500.

Combined gel permeation HPLC and reverse-phase HPLC t ol lowed by

rad i oi mi noassay have been used to separate and quantitate beta

endorphin related pepides [23. Initially, the peptides are separted by

molecular weight on gel permeation HPLC under denaturing conditions C 6

M

guanidine HC1/0.2 M triethylamine phosphate!. This system provided

accurate molecular weight detemi nati ons tor proteins ot 500 - 20,000

da It on s. Since larger proteins eluted in the void volume, it is not

possiblle to determine their molecular weights. These beta endorphin

related peptides were then analyzed by reverse-phase HPLC employing

acetonitrile as the organic moditier and either tritluoroacetic acid

[ T F A 3 or heptat 1 uor obutyr i c acid CHFBA3 as the ion pairing agent.

Separation of the peptides by reverse-phase HPLC revealed small

differences in their structures resulting from minor post-translational

modifications , such as acetylation, which affect peptide

hydrohob i c i ty . A combination of gel permeation and reverse phase HPLC

were also used by Anderson and Mole [853 to isolate chemical and

enzymatic cleavage fragments of plasma amyloid P-component for amino

79
acid sequence analysis.

4.3 Reverse-phase High Performance Liquid Chromatography

Peptides and protein of molecular weight less than 12,000 can be

separated and eluted from reverse phase columns under the appropriate

conditions of buffer composition, pH and organic modifiers. Reverse

phase simply means that the molbile phase is more polar than the

effective stationary phase. These reversed phase chromatography

columns are prepared by binding alkyl residues to inorganic support

materials; both octyl and octyldecyl-bonded as well as alkylphenyl,

phenyl, and cyanoalkyl phases have been used successfully for peptide

separations [70,861. The chemical composition of the stationary phase

is a function of unreacted surface available to the mobile phase

components. Recently, Tempst and coworkers [871 found cyanopr opy 1 si 1 yl

columns are able to separate peptides ranging in size from 5 amino

acids to 68 kilodaltons.

The elution of peptides from the column requires an organic solvent

which must be an effective solvent for the peptides of interest. If the peptides are not soluble in the chosen solvent, they may then

precipitate in the column. The starting solvents [acid-basic buffers]

that have been used to separate peptides by reverse-phase HPLC are dilute phosphoric acid [881, high concentrations of acetic acid or formic acid [891 and low concentrations of perflourinated organic

acids, particularly tr i f 1 our oacet i c acid [901. The main limiting

solvents used in these systems include 1-propanol, 2-propanol, ethanol,

methanol, or acetonitrile. Hermodson suggested separating small and

hydrohilic peptides with methanol or acetonitrile gradients and larger

hydrophobic peptides with propanol gradients [911. All of these

solvents allow spectrophoto metric detection of the peptide bond in the

range of 200 to 220 nm , hence allowing monitoring at high sensitivity.

Other than insolubility of the peptide in the organic solvents, these

solvents do present other problems. For example, nonvolatile solvents

such as phosphoric acid would be difficult to remove from the peptide

80
mixture. Also, the high viscosity of some of the solvents such as propanol may limit resolution but usually has no effect on peptide

recovery [91]. And finally, since these peptides are partially

denatured in the solvents therefore resulting in loss of biological

activity, separation of enzymes on these columns may prove to be

difficult.

In the presence of strongly ultraviolet light absorbing solvents such

as pyridine, nanogram levels of peptides can be detected by postcolumn

derivitization of free amino groups with fluorescamine [92] or the

epsilon amino group of lysine with 0-phthal adehyde [931. Meek has also

shown that deri vatizing peptides with 3,6 di ni trophthal i c anhydride and

2- carboxy-4,6-dinitrophthalic anhydride allows analysis of peptides in

tissue extracts that are in too low concentrations [< 100 pmol] to be

detected by absorbance at 210 nm [94],

The pore size of a reverse-phase HPLC column dramatically affects the

behavior of the peptides on the column [95]. If peptides less than 25

residues are to be resolved there are high density columns available

with small pore sizes [97] that will adequately resolve these small

peptides. Many researchers claim that neither flow rate, length of the

column, nor temperature [96] has a dramatic effect on the resolution of

peptides on reverse phase HPLC. Rivier [97] claims higher temperatures

resulted in lower resolution but other investigators claim temperatures

of 45°C result in better separations [98] or where the effect of

temperature is of minor importance [99]. Although sample loading does

affect retention of peptides with alkylsilicas, overloading does not

result in total loss of resolution as may occur in size exclusion of

ion-exchange chromatography [100].

Peptides tend to give better resolution on alkylsilicas for solvent pH

values in the range of 2-3 [100]. When choosing a solvent pH, an investigator must take into account the possibility that a specific

protein will be insoluble at or near its pi value.

An advantage of reverse-phase HPLC is that very closely related

molecules can be separated using combinations of pH and pairing ion

81
modulation under specific gradient elution conditions [88,100,1011.

Proper choice of a charged counter ion depends on the composition and

sequence of the peptide [1021. Hydrophobic ion-pairing reagents can be

used to significantly increase retention of polar peptides whereas

hydrophilic ion pairs increase the polarity of the peptide resulting in

a decrease in its retention on a re versed-phase column. This technique

offers many other advantages: the chromatography is fast; detection of

the peptide bond at 210 nm is quantitative and highly sensitive; and

recovery of the peptides loaded on the column is complete.

The major application of revers-phase HPLC is the isolation and

purification of peptides C 1031 . Other than giving an indication of the

hydropho bicity of the peptides , limited information about the peptide

is obtained by this method. In addition, reverse phase HPLC has been

used as a procedure to detect protein variants [104,105,106,1071; for

analytical peptide mapping [108,1091; as well as for analysis of

synthetic peptides produced by solid-phase or solution peptide

synthesis C1101. Reverse-phase HPLC has also facilitated structural

studies of glycoproteins [1111.

5 conclusion

Paper chromatography and electrophoresis were among the first methods

used to separate peptides from complex mixtures. Thin layer techniques

were latter adapted for their greater capacity, and preparative

potential. Both the paper and thin layer techniques have proven to be

useful in qualitative analysis but limited in their quantitative

abilities. They are also unable to separate larger peptides, peptides

which are insoluble in the required buffers, and samples which contain

high concentrations of inorganic ions. At present time polyacrylamide

gel electrophoresis is limited in its ability to deal with mixtures

containing peptides of widely ranging sizes and the small peptides. The

two main problems with this technique are: co-electrophoresis of small

peptides and limitations in the ability to detect the small peptides,

82
due to diffusion and loss of the peptides from the gel. High

performance liquid chromatography, a popular method of choice for

studying peptides offers the capability of: estimating of the molecular

weight of small peptides, through the use of size exclusion

chromatography; information on the charge of the peptides, by the use

of ion exchange chromatography; and a measure of peptide hydrophobicity

may be determined by reverse-phase chromatography. Although all these

procedures allow us to study unique problems of peptides, there is not

yet available a general analytical technique for the separation and

detection of peptides with widely varying molecular weights,

particularly for the small molecular weight peptides containing less

than i 0 amino acid residues.

This nor k was supported by the National Institute of Mental Health and

the Donald and Char it y Yarborough Fund for Medical Research.

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[95] Van der Rest, M. , Bennett, H.P.J., Solomon, S. and Glorieux,

F.H., 5 iochesical J. 1930, 191, 253-256.

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104, Academic Press, New York 1984 pp. 190-212.

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1932, pp’47-54.

87
VIRUSES AND VIRAL COMPONENTS IN EXTRACTS

OF INFECTED CELLS i ANALYSIS BY AGAROSE

GEL ELECTROPHORESIS

Philip Serwer

Department of Biochemistry

The University of Texas Health Science Center

San Antonio, TX 78284-7760

Summary

Detection, isolation, chracterizat ion and quantitation of viruses

and related particles are goals of diagnostic and basic virology.

Progress in the use of agarose gel electrophoresis to accomplish

these goals is described here. This technique is comparatively inexpensive

and nonselective for the detection of particles in either unfractionated

or partially-fractionated lysates of infected cells. The size of

spherical particles has been measured with an error of ± 17= by two-dimensional

agarose gel electrophoresis. Spheres can be discriminated from rods

by measurement of electrophoretic mobility (p) as a function of gel

concentration. Values of solid support-free p can be measured.

Agarose gel electrophoresis is used in the analysis of bacteriophage

assembly pathways and has potential for use in analysis and diagnosis

of virus-caused diseases.

88
1 . Introduction

Detection and characterization of viruses and subviral, virusrelated particles are objectives of diagnostic virology (reviewed

in 1-3) and basic virology (reviewed in 4-6). Detection has been

performed by testing for particles with either the known biological,

biochemical or immunological properties of known viruses and virusrelated particles. However, specific biological, biochemical and

immunological properties, in contrast to physical properties, are

not a characteristic of all virus-like particles. Therefore, testing

for particles with known biological, biochemical and immunological

properties will be referred to as intrinsically selective. To eliminate intrinsic selectivity, detection has been performed by testing

for particles with virus-like physical properties. For example,

in clinical virology, electron microscopy has been used to detect

viruses (7-9). In basic virology, viruses and viral precursors have

been detected by electron microscopy, rate zonal centrifugation,

buoyant density centrifugation and gel electrophoresis (reviewed

in 4-7,10,11). Although there is no intrinsic selectivity in the

use of such physical procedures, these procedures can be selective

(extrinsic selectivity) because of: a) alteration of the particles

by the procedure used for their analysis (for example, either disintegration of particles or adherence of particles to solid supports),

b) failure of the procedure of analysis to discriminate two related,

but different, particles.

Gel electrophoresis is 10-100 times less expensive (in time

and cost) than the other physical procedures above. Procedures for

measuring electrophoretic mobility (p) ± 2% in agarose gels have

89
been developed (12). Therefore, it appears worthwhile to explore

the possibilities for use of agarose gel electrophoresis in basic

and clinical virology. Agarose gels with pore exclusion limits as

large as 400 nm and as small as 10 nm in radius can be used for electro

phoresis (P. Serwer and S. J. Hayes, unpublished data). Therefore,

pore size is not a limit for the analysis of known viruses.

2 . Adherence to the Gel

In some cases, adherence to agarose has limited the use of agarose

gel electrophoresis for the detection and characterization of viruses

and virus-related particles. Adherence has been revealed by: a) an

increase in the sharpness of bands as the concentration of sample

is increased (13), and b) a decrease in the |y| to 0 as time of electro

phoresis is increased (14). Bacteriophage T7 , but not the genetically

related bacteriophage T3 , adheres detectably to agarose during electrophoresis. Fibers on an external tail of T7 are required for both

attachment to the bacterial host and adherence to agarose (13,15).

Although other double-stranded DNA bacteriophages have tail fibers

(16), only T7 among <629, P22, T3 , T4 and T5 adheres detectably to

agarose during electrophoresis (reviewed in [6]). A variety of other

bacterial, plant and animal viruses also appear not to adhere to

agarose during electrophoresis (reviewed in [6]). At present, the

structure of viral components is not well enough known to understand

the cause and predict the occurrence of such adherence.

Agarose is a mixture of related polymers and characteristics

of agarose gels can vary with the source and preparation of agarose.

In addition, the properties of agarose gels can be modified by either

covalently modifying agarose or by adding other polymers to agarose

90
before gelation (reviewed in [17]). Thus, if a particle adheres

to gels made from one preparation of agarose, it appears worthwhile

to test additional preparations in order to find a preparation that

is nonadherent. One possible procedure for performing this test

has previously been described (13). Most commercially-prepared agarose

and mixtures containing agarose are sold with a manufacturer-determined

index of electro-osmosis (EEO) , the electrophoresis-induced movement

of buffer through the gel (18,19). EEO increases as the concentration

of anionic groups on agarose polymers increases (20). As agarose

is increasingly used for the electrophoresis of proteins and viruses,

it is hoped that eventually assays of the protein binding of agarose

gels will be developed and the results of such assays will be provided

by the manufacturer of commercially-produced agarose.

3 . Detection of Particles

Viruses consist of nucleic acid packaged within a container

(capsid) of protein that in some viruses is covered by pieces of

cellular membrane (21). Because the nucleic acid is almost always

resistant to nucleases, agarose gel electrophoresis of nucleasetreated samples, followed by staining for nucleic acid and for protein,

is a comparatively non-selective procedure for detecting either viruses

or virus-related nucleoproteins . Ethidium bromide has been used

to stain DNA packaged in bacteriophages, without staining protein

(13,22). The limits of detection were 2 ng of DNA (= 1.0 x 10® particles

of bacteriophage <f> 29, molecular weight of DNA = 12 x 10® or 1.0 x 10^

particles of bacteriophage T4 , molecular weight of DNA = 115 x 10®).

The data (23) suggest that improved sensitivity of DNA-specific staining

is possible. A systematic attempt to increase the staining sensitivity

for nucleic acids in agarose gels should be made.

91
Agarose gel electrophoresis, followed by staining with ethidium

bromide, has been used to detect viruses in unfractionated extracts

of cells infected by a tail fiberless mutant of bacteriophage T7

(24), panicum mosaic virus (a plant virus that infects grasses) and

viruses related to panicum mosaic virus (25). This procedure should

be applicable for diagnosis of the diseases of both plants and animals.

Among possible limitations of the above procedure are: a) sensitivity,

b) possible adherence of viruses to agarose gels (see the Discussion

above), and c) interference by cellular components. Thus far, interference

by cellular components has not been a limitation. A procedure for

assisting in isolating viruses from cellular components during agarose

gel electrophoresis is described below.

4 . Characterization of Particles

4.1. Staining

In addition to its use for detection in agarose gels,

nucleic acid-specific staining can also be used to characterize the

internal contents of viruses and related particles after fractionation

by agarose gel electrophoresis. For instance, a staining procedure

for detecting bacteriophage capsids with subgenomic, packaged DNA

was developed and used for bacteriophage P22 (26). Because of their

comparatively small amount, the finding of such particles was like

finding a needle in a haystack. By staining of the viral nucleic

acid with acridine dyes it should also be possible to distinguish

DNA-containing from RNA-containing viruses after electrophoresis (27).

4 .

2

Electron Microscopy

To help characterize particles after detection by agarose

gel electrophoresis of either unfractionated or partially fractionated

92
extracts, these particles are eluted from the gel and examined by

electron microscopy. For instance, in the case of tobacco mosaic

virus (TMV), a rod-shaped plant virus with a length of 300 nm and

a radius of 9 nm (28), two sharp bands, and possibly a third, were

observed by Coomassie blue staining of agarose gels after electrophoresis of a partially fractionated extract of infected Hicks tobacco

leaves (Fig. 1). These bands can also be seen, without significant

loss in clarity, in profiles of unfractionated extracts less than

two weeks old (not shown). To determine the characteristics of the

particle that forms each of these bands, the following procedure

(not previously described) was used to transfer particles directly

from the gel to a support film for electron microscopy.

Two identical samples were separately subjected to electrophoresis

in a 0.9% agarose slab as shown in Figure 1. The region of the gel

traversed by one of the two samples was excised and stained with

Coomassie blue. Using this stained section of the gel as a template,

the position of the above bands in the unstained section of the gel

was determined. A groove was cut through the estimated center of

each band, using either a spatula or a razor knife. Into each groove

was placed a 400 mesh, copper electron microscope grid, covered with

a carbon film. The carbon film had been placed on the grid, as previously described (29), and faced the cathode. To help deposit particles

on the carbon film, electrophoresis at 1 V/cm was then performed

for 2 hr with the grids in the gel. The grids were then removed

from the gel and negatively stained with uranyl acetate, as previously

described (29). To determine whether or not the grids had been accurately

placed for elution, the gel used for elution was subsequently stained

with Coomassie blue.

93
Rods with the radius and central hole of TMV were observed when

particles eluted from the region of the gel containing band 2 of

Figure 1 were observed (Fig. 2). However, 34 of 52 particles observed

were too long to be monomers and had the length of an end-to-end

TMV dimer. A gap at the point of joining between monomers was observed

at the mid-point of some dimers (arrowhead in Fig. 2). By a similar

analysis, the band labeled 3 in Fig. 1 is formed by an end-to-end

trimer of TMV. A band potentially formed by TMV monomers is indicated

with a question mark in Figure 1. End-to-end multimerization of

TMV has previously been observed by rate zonal centrifugation (30).

4 . 3 Sieving during unidirectional electrophoresis

The electrophoretic mobility (y) of a particle in an agarose

gel is determined by the: a) y in the absence of the gel (y Q

) and

b) reduction of |y| caused by collision of the particle with the

gel's network of fibres (sieving). For virus-sized (13-42 nm in

radius) spherical particles in gels with an agarose percentage (A)

_< 0.9, the following relationship has been found (reviewed in [17]):

y = (yQ + yE

) e

" KR-A (1)

yE describes the EEO and Kr is the empirically-obtained slope of

a semilogarithmic y vs. A_ plot. Kr increases as the radius (R) increases

(17). For A J> 0.9, semilogarithmic y vs. A^ plots have significant

nonlinearity and Kr progressively increases as A increases (convex

curvature )

.

In the presence of spherical size standards of known R, the

R of a spherical particle can be determined from the Kr with an accuracy

of ± 8% (reviewed in [17]). By plotting y vs. A (instead of In j u

1

vs. A) it has been found for at least one 6.5% hydroxyethylated agarose

94
that A extrapolated to a p of 0 (A0

) is independent of p0 and decreases

as R increases (31). From A.0 , R can be determined with an accuracy

of ± 470 (31).

Semilogarithmic p vs. A plots for rod-shaped viruses are linear

for A below a critical value that decreases as the length of the

rod increases (32). Above this critical _A the plot becomes concave

(32), in contrast to the convex curvature observed for spheres (above).

Thus, a rod-shaped virus can be discriminated from a spherical virus

by semilogarithmic p vs. _A plots. Additional studies are needed

to determine how accurately the shape of a particle can be determined

by measurement of its sieving in agarose gels, without additional

information

.

4 . 4 Two-dimensional electrophoresis

After a single, unidirectional electrophoresis, the position

of a particle in a gel is a function of two independent properties

of the particle, p0 and a size-shape-describing parameter (Kr or R for

a sphere, if A < 0.9). Position in the gel does not uniquely determine

pQ and R. To obtain a gel profile with a one-to-one correspondence

between position in the gel and the R of a spherical particle, the

following procedure of electrophoresis in two directions (two-dimensional

agarose gel electrophoresis) was developed.

A first electrophoresis is performed in a comparatively dilute

agarose gel (first-dimension gel). Subsequently, particles fractionated

in the first-dimension gel are subjected to electrophoresis orthogonal

to the first-dimension gel and migrate into a more concentrated gel

(second-dimension gel) adjacent to the first dimension gel. As previously

shown (33), two-dimensional agarose gel electrophoresis distributes

95
all spherical particles of any one R along a straight line (size

line) that passes through the effective origin of electrophoresis

and makes an angle, 0, with the first-dimension gel. As R decreases,

0 increases and R(0) is determined with size standards (33). If

the _A of the first-dimension gel is low enough so that its sieving

is too low to be detected, then the pQ

1 of a particle after two-dimensional

agarose gel electrophoresis is p in the first-dimension gel.

Because the position of a spherical particle after two-dimensional

agarose gel electrophoresis uniquely determines R and p Q , the patterns

obtained by this procedure can, in theory, be used to determine the

distribution of R and pQ for heterogeneous particles in a mixture.

The automated densitometry and computer software for achieving this

goal has not yet been developed. The sorting by size that occurs

during two-dimensional agarose gel electrophoresis should also help

separate viruses from interfering subcellular fragments of cells,

during the fractionation of crude extracts of virus-infected cells.

By adjusting the A_ of the second-dimension gel, resolution of R as

good as ± 0.3 nm has been achieved for particles approximately 30

nm in R (33). Thus, in the presence of standards of known R, R can

be measured ± 17, by two-dimensional agarose gel electrophoresis.

5 . Analysis of Viral Assembly Pathways

When analyzing the assembly pathway of a virus, one first tries

to detect particles (called assembly intermediates) in the pathway.

After detecting potential assembly intermediates, their kinetics

of appearance is determined (examples are in 4-6, 34). Determination

of these kinetics requires quantitation of potential intermediates

in multiple samples. Performing this quantitation by agarose gel

electrophoresis, instead of either centrifugation or electron microscopy,

96
converts work requiring several months into work that can be (and

has been) completed in a week (a comparatively recent example is

in [34]). The data obtained are also usually more accurate than

the data obtained by centrifugation.

If agarose gel electrophoresis is not, by itself, sufficient

to fractionate all potential assembly intermediates, agarose gel

electrophoresis can be used as the second step in a two-step procedure

of detection and quantitation (6). The first step can be centrifugation

(either rate zonal or buoyant density), electrophoresis (either density

gradient or gel; see above), or column chromatography.

6 . Determining the Cause of Infectious Disease: Analogy with Determining

of Viral Assembly Pathways

If the virus that causes an infectious disease is the most abundant

virus in extracts of infected tissue, it is comparatively easy to

identify this virus. However, if other, non-causative viruses or

virus-like particles (secondary invaders, for instance) are more

abundant than the particle that causes the disease, identification

of the particle that causes the disease becomes more difficult. The

difficulties are compounded if the virus has not been biologically

cloned and propagated so that Koch's second and third postulates

can't be used. Finding a disease-causing particle, in the presence

of a larger amount of other particles, is analogous to the finding

of an assembly intermediate that is present in comparatively small

amounts. The success of agarose gel electrophoresis-ethidium staining

in this latter pursuit (above) suggests that it is worthwhile to

use this procedure in attempts to detect, isolate and characterize

disease-causing viruses.

97
If a potential disease-causing particle is detected by agarose

gel electrophoresis, comparison of the particle's appearance kinetics

with the kinetics of disease symptoms is a procedure to help determine

the relationship of symptoms to the presence of the particle. Experimentally,

the particle's appearance kinetics could be determined by agarose

gel electrophoresis, as performed for determining the appearance

kinetics of assembly intermediates (above). Extending the analogy

with assembly even further, it is possible that some diseases of

currently unknown cause are caused by more than one virus, the different

viruses appearing in a definable temporal order. Instead of determining

the single virus that causes a disease, in this case one would determine

the viral pathway of causation. For example, evidence of a bacterial

pathway of causation has been presented for early-onset periodontitis

( 35 ).

98
Acknowledgements

I thank Gerald Merrill and Elena T. Moreno for performing the

experiment in Fig. 1, Paul Horowitz for reviewing a draft of this

manuscript, and Anna Uriegas for typing the manuscript. Work done

in the author's laboratory was supported by grants from the National

Institutes of Health (GM 24365 and AI 22568) and the Robert A. Welch

Foundation (AQ-764).

99
References

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Haven 1982.

2. Smith, T.F., in: Washington, J.A. (Ed.) "Laboratory Procedures

in Clinical Microbiology", Springer-Verlag , New York 1985, pp. 537-

624.

3. Kucera, L.S. and Myrvik, Q.N. "Fundamentals of Medical Virology:

For Students of Medicine and Related Sciences", Lea and Feabiger,

Philadelphia 1985.

4. Kellenberger , E., Biosystems 1980, 12 , 201-223

.

5. Georgopoulos , C., Tilly, K. and Casjens, S., in Hendrix, R.W.

,

Roberts, J.W., Stahl, F.W. and Weisberg, R.A. (Eds.) "Lambda IT",

Cold Spring Harbor 1983, pp . 279-304.

6. Serwer, P., ASM News 1984, 50 ,414-417

.

7. Francki, R.I.B., in Kado, C.I. and Agrawal, H.O. (Eds.) "Principles

and Techniques in Plant Virology," Van Nostrand Reinhold, New York

1972, pp. 295-335.

8. Almeida, J.D., in Bachmann, P.A. (Ed.) "Current Topics in Microbiology and Immunology", Vol. 104, Springer-Verlag, Berlin 1983,

pp. 147-158.

9. Madeley, C.R., in de la Maza, L.M. and Peterson, E.M. (Eds.)

"Medical Virology II", Elsevier Biomedical, New York 1983, pp. 81-

109.

10. Poison, A. and Russell, B., in Maramorosch, K. and Koprowski, H. (Eds.) "Methods in Virology", Vol. II, Academic Press, New

York 1967, pp . 391-426.

11. Anderson, N.G., in Bachmann, P.A. (Ed.) "Current Topics in

Microbiology and Immunology," Vol. 104, Springer-Verlag, Berlin

1983, pp. 197-217.

12. Serwer, P., Electrophoresis 1983, 4_, 227-231.

13. Serwer, P. and Hayes, S.J., Electrophoresis 1982, ^,76-80.

14. Serwer, P., Watson, R.H. and Hayes, S.J., J. Virol. 1982, 42 , 583-594.

15. Serwer, P., Watson, R.H. , Hayes, S.J. and Allen, J.L., J. Mol. Biol.

170,447-469.

16.

Wood, W.B. and King, J., in Fraenkel-Conrat , H. and Wagner, R.R.

(Eds.) "Comprehensive Virology", Vol. 13, Plenum, New York 1979 , pp. 581-633.

100
17. Serwer, P., Electrophoresis 1983, 9^,375-382.

18. Quast, R. , J. Chromatogr. 1971, 59,905-912.

19. Serwer, P. and Hayes, S.J., Electrophoresis _3, 80-85.

20. Cook, R.B., in Allen, R.C. and Arnaud , P. (Eds.) "Electrophoresis

'81", de Gruyter, Berlin 1981, pp. 219-228.

21. Luria, S.E., Darnell, J.E., Jr., Baltimore, D. and Campbell, A.,

"General Virology", Wiley, New York 1978.

22. Griess, G.A. , Serwer, P., and Horowitz, P.M., Biopolymers 1985,

_29, 1635-1696.

23. Nairn, R.S., Dodson, M.L. and Humphrey, R.M. , Biochem. Biophys.

Methods 1982, _6, 95-103.

29.

Serwer, P. , Seanotes 1982 (A commercial publication of the Marine

Colloids Division of the FMC Corporation), Autumn edition.

25. Serwer, P., Moreno, E.T., Hayes, S.J., Berger, P., Langham, M.

and Toler, R.W. , Electrophoresis 1989, _5, 202-208.

26. Serwer, P. and Gope , R. , J. Virol. 1989, 99 , 293-296

.

27. Mayor, D.M. , in Richter, G.W. and Epstein, M.A. (Eds.) "Intern.

Rev. Exptl. Pathol.", Vol. 2, Academic Press, New York 1963,

pp . 1-93.

28. Klug, A. and Caspar, D.L.D., Adv. Virus Res. 1960, _7, 225-325.

29. Serwer, P., J. Mol. Biol. 1976, 107,271-291.

30. Francki, R.I.B., Virology 1966, 30 , 388-396

.

31. Serwer, P. and Allen, J.L., Electrophoresis 1983, _9, 273-276.

32. Serwer, P., Merrill, G. , Moreno, E.T. and Herrmann, R. , in Peeters, H.

(Ed.) "Protides of the Biological Fluids", Vol. 33, Pergamon, Oxford

1985, pp. 517-520.

33. Serwer, P., Hayes, S.J. and Griess, G.A., Anal. Biochem. 1986,

in press.

39. Serwer, P. and Watson, R.H., Virology 1985, 190 , 80-90

.

35. Williams, B.L., Ebersole, J.L., Spektor, M.D. and Page, R.C.,

Infect. Immun. 1985, 99,792-750.

101
Figure 1. Agarose gel electrophoresis of an unfractionated extract of leaves infected

by TMV. Thirty g of Hicks tobacco leaves infected with the common strain

of TMV, received from M. Langham and R.W. Toler, Texas A & M University,

were mixed with 100 ml of ice-cold 0.05 M sodium phosphate, pH 7.4, 0.001

M MgCl 2 (electrophoresis buffer) and were subsequently ground in a Waring

blender for 5 min. This extract of ground leaves was clarified by two

successive centrifugations at 10,000 rpm, 4°C for 10 min in a Beckman JA-14

rotor. Particles of TMV were pelleted by centrifugation at 18,000 rpm,

4°C for 16 hr. in a Beckman JA-20 rotor. After resuspending the pellet

in a 1/100X volume of electrophoresis buffer, the preparation of virus

was clarified once more, as described above. To 2 pi this crude preparation

of TMV was added: 30 pi of electrophoresis buffer; 1 pi of a preparation

containing 3 pg of the spherical RNA bacteriophage, MS-2 (used as a marker

for p); and 62 pi of 4% sucrose, 400 pg/ml bromphenol blue in electrophoresis

buffer. Of this mixture, 50 pi was subjected to electrophoresis in a 0.97,

agarose gel (Seakem ME agarose, purchased from the Marine Colloids Division

of the FMC Corporation) at 1 V/cm, room temperature (25 ± 3°C) for 16 hr.

The 0.9% gel was embedded in a 1 . 570 agarose frame, as described in ref.

19. After electrophoresis, protein in the gel was stained with Coomassie

blue (19). The origin of electrophoresis is indicated by the arrowheads;

the direction of electrophoresis (toward the anode) is indicated by the arrow. Bands determined by electron microscopy (see the text) to be those

of TMV dimers and trimers are indicated by 2 and 3, respectively. The

band formed by bacteriophage MS-2 is indicated.

-3

-2

-I?

-MS-2

i

!

t02
Figure 2. Electron microscopy of a dimer of TMV. Particles from

the region of an agarose gel indicated by 2 in Fig. 1 were eluted from the gel and prepared by electron microscopy,

as described in the text. An example of a dimeric TMV

rod is shown; the point of joining between the two monomers

of the dimer is indicated by the arrowhead. The arrow

indicates a monomeric TMV rod.

-vV

‘

' m

200 nm

103
Gel Electrophoresis of DNA

Nancy C. Stellwagen

Department of Biochemistry

University of Iowa

Iowa City, Iowa 52242

Recent studies of the electrophoresis of DNA in agarose and in polyacrylamide gels are summarized. For agarose gel electrophoresis, the

topics covered include: quantitative studies of the electrophoresis of

linear restriction fragments and spherical DNA molecules; the use of

pulsed electric fields; the separation of chromosome-sized molecules

of DNA using alternating inhomogeneous electric fields; the application of electric birefringence to the study of gel electrophoresis;

recent developments in reptation theory; the use of gel electrophoresis

to study conformational transitions; the pore size and fiber radius of

various types of agarose; and recent advances in experimental techniques. For polyacrylamide gel electrophoresis, the topics covered

include: quantitative studies of the electrophoresis of linear restriction fragments; the anomalous mobility exhibited by some restriction

fragments; the use of denaturing gradient gels to study single base

mutations; the study of protein-DNA interactions by a gel electrophoresis assay; and improvements in experimental procedures.

104
1 Introduction

Electrophoresis is the movement of charged particles suspended in a

medium under the influence of an electric field (1, 2). In free

solution, the electrophoretic mobility of DNA molecules is independent

of molecular weight (3), because the linear charge density is constant

for all molecules. However, when electrophoresis takes place in a

supporting gel medium, molecular weight separation occurs. Various

applications of gel electrophoresis range from the separation of DNA

fragments containing a few base pairs for sequence analysis, to the

separation of whole chromosomes, containing hundreds of thousands of

base pairs (4).

Despite the importance of gel electrophoresis in the development of

modern molecular biology, the actual mechanism of the molecular weight

separation of DNA fragments in the gel is not clear (5, 6). Two

different mechanisms have been proposed. The first, based on the

Ogston model of a random meshwork of fibers (7), hypothesizes that the

electrophoretic mobility of a macromolecule is proportional to the

volume fraction of the pores of a gel that it can enter (5, 8-10).

Since the average pore size decreases with increasing gel concentration, (5, 8-11), electrophoretic mobility is expected to decrease

with increasing gel concentration. The second mechanism is based on

reptation, the "worm-like" or "snake-like", end-on migration of DNA

molecules through the pores of gel (12-14). This mechanism has been

used to explain the inverse relationship between electrophoretic

mobility and DNA molecular weight observed experimentally (15).

105
In this review, recent experiments utilizing gel electrophoresis will

be summarized. The emphasis will be on illustrating the different

types of experiments for which gel electrophoresis is a useful technique, rather than an exhaustive survey of the literature. Two types

of gel matrices are commonly used for the gel electrophoresis of DNA:

agarose and polyacrylamide. Since different types of experiments are

carried out in the two media, they will be presented separately. Some

practical aspects of gel electrophoresis are discussed in (16-18).

2 Agarose gel el ectrophoresis

2.1 Background

The chemistry of agarose and the physical properties of agarose gels

have been reviewed by Serwer (19). Solid agarose is dissolved by

heating in an appropriate buffer, and undergoes gelation upon cooling.

Gelation in aqueous solution is accompanied by the formation of aggregates of double-stranded helices (20, 21). Gels formed in the presence of salt have larger void spaces and a stronger tendency for

sections of rods to group together (21). Aqueous solutions of agarose

appear to undergo a sol-sol transition as the first step in gelation

(22). Under conditions where gelation occurs over a period of weeks

to months, static and dynamic light scattering experiments indicate

the presence of a transition in the sol state which occurs after a

characteristic incubation time (22). This transition may reflect the

coi 1 - to- he 1 ix transition preceding gelation (22).

106
2.2 Linear DNA restriction fragments

2.

2.

a Relation between mobility and molecular weight

Several recent studies have focused on the quantitative relationship

between gel electrophoretic mobility and DNA molecular weight. For

high molecular weight fragments, these curves depend on experimental

parameters such as gel concentration and electric field strength (6,

23). Southern (15) proposed that the reciprocal of electrophoretic

mobility is linearly related to molecular weight. Bearden (24) pro-

-2/3

posed that gel electrophoretic mobilities are related to M , over a

restricted range of experimental conditions. Stellwagen (25) proposed

that mobilities of restriction fragments containing fewer than 1000

base pairs (bp) are linearly correlated with M ' , because of the

variation of the "hydrodynamic surface area" of fragments of this size

with molecular weight. Edmondson and Gary (26) proposed that gel

— y mobilities are related to [Mi(L 1 /L0 ) - M2 ], where Mi and L 0 are

constants and x and M2 depend on the gel concentration.

2.2.

b Determination of

The negative slope of the natural logarithm of the apparent mobility

as a function of gel concentration is called the retardation coefficient (5, 8-10). A semi-logarithmic plot of the apparent mobility as

a function of gel concentration is linear for DNA restriction fragments up to about 1-3 kilobase pairs (kbp) in size (5, 6, 24-28). For

107
larger fragments such plots show a concave upward curvature (6, 24,

25, 27, 28). The degree of curvature depends on the DNA molecular

weight and applied voltage, and can be essentially eliminated by

extrapolating the mobilities observed at each gel concentration to

zero electric field strength (6). These effects are due to the fact

that the mobilities of restriction fragments in agarose gels increase

linearly with increasing electric field strength; the rate of increase

decreases with increasing molecular weight (6). It has been suggested

that high electric fields may not give the particles enough time to

accelerate to their free solution electrophoretic velocities before

colliding with a gel fiber (29).

According to pore size distribution theory (5, 8-10), the retardation

coefficients can be related to molecular parameters by

K

r 1/2 = a + b ( R+r ) (1)

where a and b are empirically determined constants, R is the radius of

a spherical particle moving through the gel, and r is the gel fiber

radius. For highly asymmetric particles, such as small DNA restriction fragments, the correct value to use for R has been a matter of

debate (5). Bearden (24) used a prolate ellipsoid of revolution with

constant axial ratio. Stellwagen used the hydrodynamic surface area

determined from electric birefringence experiments for restriction

fragments < 1 kbp (25), and the root-mean-square (rms) radius of

gyration for larger fragments (6). Serwer and Allen (28) used an

effective hydrodynamic radius calculated from the radius of gyration.

Edmonson and Gray (26) approximated the dependence of on macro10B
molecular length by a logarithmic function. A log-log plot of vs.

0 6 molecular weight shows that ^ M for DNA fragments between 0.5

and 12 kbp in size, if the Kp values are determined from mobilities

extrapolated to zero electric field strength (6).

2.2.

c Apparent mobility at zero gel concentration

From semilogarithmic plots of mobility as a function of gel concentration, estimates can be made of the mobility of DNA fragments at

zero gel concentration. This mobility should be close to the free

solution value (8). A summary of the literature values of the

mobility at zero gel concentration is given in Table I.

2.3 Spherical DNA particles

2.

3.

a Mobility as a function of molecular weight

In HGT agarose, (trademark name of Marine Colloids) open circle

(relaxed) and closed circle (supercoi led) DNA molecules were found to

have the same mobility at zero gel concentration as linear DNA molecules of the same molecular weight (28). Careful control of temperature and applied voltage were required to obtain this result (28).

A plot of the logarithm of the mobility of spherical bacteriophage

particles as a function of gel concentration was found to be concave

downward at high gel concentrations (28, 32, 33), opposite to the

curvature observed with linear DNA molecules. In SeaPlaque agarose

109
(trademark name of Marine Colloids), the mobility of bacteriophage and

related particles was found to be linearly dependent on gel concentration (34). The intercept of this plot with the axis of zero gel

.concentration was found to be related to the radius of the bacteriophage particle. 2.3.

b Superhelix density

One application of agarose gel electrophoresis is the determination of

superhelix density of closed circle DNA molecules. A series of topological isomers differing by one in linking number can be clearly

resolved on agarose gels (35, 36). Typical applications have ranged

from determining the helical periodicity of different conformations of

DNA (37), to evaluating the dependence of conformational transitions

on superhelix density (38).

2.3.

c Two-dimensional electrophoresis

A variant of the method of determining superhelix density is running

the electrophoresis in each of two perpendicular directions, using the

same electric field strength in each direction. Before electrophoresis

in the second direction, the gel is soaked in an intercalating agent

(39). Topoisomers are resolved into distinct spots that lie along a

smooth curve, each spot differing by one in linking number (39). An

abrupt shift in the curve is an indication of a conformational transition. Current applications of this technique include determining

helical periodicity (39), cruciform formation (40), and the _i_n v i vo

occurrence of Z DNA (41).

110
Branched DNA molecules have been separated from linear molecules by

electrophoresis in two perpendicular directions, using a different

agarose concentrations and electric field strengths in each direction (42)

. The branched molecules appear on the gel diagonal, in order of

increasing molecular weight.

2.4 Pulsed field electrophoresis

2.

4.

a One dimension

Instead of a continuously applied electric field, it is possible to

use a pulsed square wave of varying amplitude and duration. The

electrophoretic pattern can be significantly varied, even though the

total energy (voltage x time) of the electric field is kept constant

(43)

. The pulse amplitude, duration, and frequency can be varied to

maximize the gel resolution in any given molecular weight range (43).

Pulse amplitudes are typically a few V/cm, applied for a few seconds

2.4.

b Two dimensions, non-uniform fields

Schwartz and Cantor (44) have devised an el ectrophoresi s procedure

using pulsed, alternating electric fields in perpendicular directions

to separate chromosome-sized molecules of DNA containing up to 2000

kbp. At least one of the perpendicular electric fields is inhomogeneous. The period and amplitudes of the applied pulses are varied

to optimize the separation in a desired molecular weight range.

Electrophoresis occurs on the diagonal of the gel (44). Another

111
experimental arrangement places the gel at a 45° angle to the mutually

perpendicular electrodes, to give a nearly linear electrophoretic

pattern in the gel (45). In these applications, the agarose gel

concentration is typically 1.0-1. 5%, with electric fields ^3 V/cm. and

switching times of 20-50 sec.

2.4.c Electric birefringence, single pulses

Electric birefringence uses refractive index differences to measure

the orientation of DNA molecules, either imbedded in a gel or in free

solution. The DNA molecules are oriented by a pulsed electric field;

the applied voltage is on the order of 1 kV/cm. , with durations in the

millisecond range. The time constant for the loss of the orientation

after removal of the electric field is characteristic of the size and

shape of the macromolecule being oriented (46). Three DNA restriction

fragments, ranging in size from 622 to 2936 bp were studied, imbedded

in agarose gels ranging in concentration from 0.2% to 1.5%. The

birefringence relaxation times of the three fragments were equal to

their free solution values if the median pore diameter of the gel was

greater than the effective hydrodynamic length of the fragment.

However, if the median pore diameter was smaller than the effective

hydrodynamic length, the birefringence relaxation times increased to

values characteristic of fully stretched DNA molecules. The apparent

elongation of the DNA molecules in the agarose gels indicates that

reptation is a likely mechanism for the el ectophoresi s of large DNA

molecules (46). These results are summarized in Table II.

112
2.4.d Electric birefringence, reversing fields

If electric field pulses which are rapidly reversed in polarity are

applied to DNA molecules imbedded in agarose gels, the birefringence

(i.e., orientation) exhibits a transient at the moment of field reversal. The time interval between the moment of field reversal and

the minimum in the orientation depends on pulse voltage, pulse length,

and DNA molecular weight (47).

2.5 Reptation theory

Lumpkin, et al. (13, 14) have presented a theory for electrophoresis

based on the axial movement, or reptation, of DNA molecules through

"tubes" in the gel. The direction of the tube is random, except for

possible bias introduced by the applied electric field. With small

electric fields, an inverse length dependence of the mobility is

found, as expected (48, 49). At higher fields the tubes become oriented,

because the field biases the direction of the leading edge of the

chain. This leads to an increase of mobility with increasing field

strength by adding a field dependent but length-independent term to

the mobility equation. Slater and Noolandi (50) have estimated that

stretching of the DNA chains would take place early in the time scale

of an electrophoresis experiment. The data of Bean and Hervet (56)

and Stellwagen (6) can be described by the equations for reptation

(14).

1 13
2.6 Structural transitions

The B/A transition in DNA has been studied by electrophoresis in

agarose gels run in an ethanol gradient (51). DNA molecules at the

mid-point of the transition appear to have the highest electrophoretic

mobility, suggesting that the B/A junction is flexible (51).

A pH dependent structural transition in the homopurine-homopyrimidine

tract in superhelical DNA has been studied by two-dimensional electrophoresis (52). A new structural conformation, based on a cruciform

structure, has been suggested (52).

2.7 Pore size and fiber radius

Three estimates have been given for the pore sizes of agarose gels of

different types. Righetti et al. (53) used polystyrene latex particles

of known diameter to determine the limiting pore size of Isogel agarose

(trademark of Marine Colloids) gels of different concentration. The

equation relating these two parameters is

P = 140.7 c"

0 ' 7

(2)

where P is the pore diameter in nm and c is the gel concentration in

percent (presumably the hydrated gel powder). Serwer and Allen (34)

studied the electrophoresis of spherical virus particles of known

molecular radius, and found the equation

P = 117.4 c (3)
for SeaPlaque agarose (trademark name of Marine Colloids). Stellwagen

(6) determined the retardation coefficients of a series of high molecular

weight DNA restriction fragments in LE agarose (trademark name of

Marine Colloids), and used the root-mean-square radius of gyration to

estimate the median pore diameter:

P = 25 + 70 c"

L0

(4)

From these three equations, the median pore diameter of a 1% gel would

be 282 nm for Isogel agarose, 234 nm for SeaPlaque agarose, and 190 nm

for LE agarose. The decreases in estimated pore size correlate with

the sieving order determined by Serwer, et al. (33) for these three

types of agarose.

The gel fiber radius of various types of agarose was determined by

1/2 Serwer, et al . (33) from plots of vs. the radius of spherical

bacteriophage particles. The fiber radii were found to be 25 nm for

HGT, ME, SeaPlaque and SeaPrep agarose, and 20 nm for Isogel agarose

(all trademark names of Marine Colloids).

2.8 Experimental improvements

Serwer (54) has described an apparatus and techniques for running

several agarose gels of different concentration simultaneously. Gels

as low as 0.035% in concentration have been formed and run (55).

Improved procedures of controlling voltage gradient and temperature

have been described (56). Multiple gels containing ethidium bromide

of different concentrations in different lanes have been used to

measure superhelix density in horizontal gels (57).

115
The electric field dependence of the apparent mobility of DNA restriction fragments has been the subject of several studies (6, 23, 25, 28,

58).

The effect of electroendosmosis on the apparent mobility observed in

different types of agarose has been discussed (6, 19, 59, 60).

An apparatus for two dimensional gel electrophoresis without gel

manipulation has been described (61).

Methods of quantifying the fluorescence of ethidium bromide-stained

horizontal gels have been developed for one-dimensional (62) and

two-dimensional (63) electrophoresis. Quantitative measurements of

f 1 uorescently stained DNA from photographic images have been described

(64). A computer-automated method of evaluating one-dimensional gel

electrophoretic radiograms has been described (65).

The resolution of DNA fragments ranging from 30 bp to 15 kbp in a

single agarose gel has been achieved by pouring a slab gel of increasing thickness from cathode to anode (66).

The effect of DNA-agarose binding affinity has been studied for several

types of agarose (67). Forward fronting of the bands at high DNA

concentrations and "displacement" by carrier DNA leading to band

sharpening are interpreted in terms of DNA-agarose interactions (67).

116
3 Polyacrylamide gel electrophoresis

3.1 Background

Polyacrylamide gels are formed by the polymerization of acrylamide and

a cross-linking agent, such as N,N' methylene bisacrylamide (Bis). The

extent of the reaction and the rate are governed by the concentration

of initiator (18). Control of the polymerization reaction depends on

reagent purity and stoichiometry (68). Small angle x-ray, neutron,

and light scattering studies (69) indicate that 8% polyacrylamide gels

polymerized with 0-0.5% Bis contain two phases, a homogeneous phase of

constant polymer content, with fiber radius increasing with increasing

Bis concentration, and an inhomogeneous phase, characterized by large

concentration fluctuations. The viscoelastic properties of 2.5% and

5% polyacrylamide gels have been measured by polarized light scattering (70).

3.2 Linear DNA restriction fragments.

3. 2. a Relation between mobility and molecular weight

The polyacrylamide gel electrophoresis of linear DNA restriction

fragments between 30 and 4000 bp in size has been studied by Stellwagen

(71). The electric field dependence of the apparent mobilities of

fragments in this size range is small (71, 72) and virtually independent

of molecular weight (71). However, for DNA molecules greater than

about 10 kbp in size, the apparent mobilties have been found to de117
crease abruptly at low electric field strengths in gels containing

1.0-1. 5% polyacrylamide and 0.5% agarose (73).

3.2.b Retardation coefficients

Stellwagen (71) determined the mobility of a series of linear restriction fragments in polyacrylamide gels of different concentrations.

The apparent mobilities observed for small DNA fragments, extrapolated

-4 2

to zero gel concentration, ranged up to 3.4 x 10 cm /V sec (71).

-4

These values are higher than the free solution mobility of 1.85 x 10

cm /V sec (3), possibly because of specific interactions between the

aromatic DNA molecules and the polyacrylamide gel (74). Stellwagen

(71) also observed that the absolute mobility of the bromphenol blue

marker dye decreased linearly with increasing polyacrylamide gel

concentration. This effect may be due to the affinity of polyacrylamide gels for aromatic compounds (74); the mobility of the marker

dye was independent of gel concentration in agarose gels (71). The

mobility of the marker dye was independent of electric field strength

in either gel medium.

Retardation coefficients, Kp, were determined for DNA restriction

fragments of different molecular weights (71). An approximately

constant value of Kp was found for fragments larger than about 800 bp

(71). For smaller fragments, Kp ^ M , which is a significantly

different molecular weight dependence than observed in agarose gels

(Kp ~ M^' ^ , see above) .

116
The values of K

R observed for the different fragments can be related

to molecular dimensions by Eq. (1), if the molecular radius is chosen

to the geometric mean radius (71). This means that the retardation

coefficients are dependent on the mass of the DNA molecules (raised to

the 1/3 power), even though these short DNA fragments are highly

asymmetric in shape. Hence molecular mass, not molecular shape,

appears to be the controlling factor in the polyacrylamide gel electrophoresis of DNA restriction fragments. It is possible that the DNA

molecules act as "magic bullets" and deform the polyacrylamide gel

during electrophoresis. Alternatively, the DNA molecules may interact

specifically with the gel matrix during electrophoresis. Both of

these processes would be expected to be mass dependent.

3.2.C Anomalous migration

When a series of linear DNA restriction fragments is electrophoresed

in a polyacrylamide gel, the mobility of a significant number of

fragments in the 300-500 bp range is found to be lower than the smooth

curve describing the mobility of the rest of the fragments (71).

Molecular weights of these anomalously migrating fragments, calculated

from their electrophoretic mobility, would be 10-20% larger than their

sequenced molecular weights (71). The anomalous mobilities are enhanced by increasing the polyacrylamide gel concentration (75), lowering the temperature of el ectrophoresi s (71, 76), or by adding Mg

++

to

the electrophoresis buffer (76). Decreasing the polyacrylamide gel

concentration (75), raising the temperature of electrophoresis (71,

76) or adding Na

+ to the electrophoresis buffer (76) has the opposite

119
effect. The anomalously migrating fragments derived from plasmid

pBR322 were found to originate from specific regions of the plasmid

(71), suggesting that a conformational feature such as bending of the

DNA helix is responsible for the anomalously slow migration. Dimerizing an anomalously migrating fragment further increases its anomalously slow migration (71).

The anomalously slow electrophoresis of some DNA fragments is particularly apparent for a kinetoplast DNA minicircle (75, 76); the molecular

weight calculated from the electrophoretic mobility is 3-4 times

larger than its sequenced molecular weight. This anomaly was attributed to a relatively sharp bend in the DNA rod (75). By making

dimers of the kinetoplast fragment and cutting with different restriction enzymes, Wu and Crothers (77) were able to prepare a series of

fragments of identical molecular weight, with the putative bend located

in different positions with respect to the ends of the fragment. The

mobilities of each of these fragments were compared and the bending

locus identified, since fragments with the bend located near the end

would be expected to have a higher mobility than fragments with the

bend located near the center.

Hagerman (78) is undertaking a systematic study of the gel electrophoretic mobilities of a series of ol igo(dA)-ol igo(dT) sequences in

order to determine the sequence dependence of the phase coherence

between sequence periodicity and helical repeat.

120
Pseudo-cruciform formation has also been shown to be responsible for

anomalously slow migration on polyacrylamide gels (79).

3.3 Denaturing gels

A large change in the electrophoretic mobility of a double-stranded

DNA molecule in a polyacrylamide gel occurs when a portion of the

molecule melts. Since melting is a cooperative phenomenon, the transition in electrophoretic mobility is abrupt if the electrophoresis

takes place in a gel containing a gradient of denaturing solvent. The

onset of melting is related to base sequence, since large DNA molecules melt in domains of different A-T content (80). Fischer, Lerman,

and coworkers (81-83) have used this technique to separate DNA fragments differing by single base-pair substitutions (83, 84). Mutations

affecting final gradient penetration lie within the first cooperatively

melting sequence. If restriction fragments of the mutant DNA molecules

are first separated in size by agarose gel electrophoresis, and then

el ectrophoresed in the perpendicular direction in a polyacrylamide gel

containing a denaturing gradient, two-dimensional separation of about

one thousand fragments is possible (85). The effects of the size of

the denatured region, the position of the denatured region in the

molecule, and the molecular weight of the undenatured portion of the

molecule on the mobility of the partially melted DNA molecule have

also been investigated (86).

Another procedure has been developed to detect single-base substitutions in DNA fragments (87), based on ribonuclease cleavage of

121
single-base mismatches in RNA:DNA heteroduplexes (87). The digestion

products are analyzed by electrophoresis in a denaturing polyacrylamide gel. This procedure has been used to detect single base mutations associated with human genetic diseases (87).

3.4 Sequencing gels

In the chemical DNA sequencing method, the DNA is end-labeled, parially cleaved at each of the four bases in four separate reactions,

and the digestion products el ectrophoresed on denaturing polyacrylamide gels (88). Several hundred nucleotides can be separated using

current electrophoresis procedures. From the nucleotide ladder produced, the base sequence of the DNA fragment can be read by inspection. Current applications of sequencing ladders, besides determining

base sequence, involve the technique of "foot-printing". In this

procedure, a ligand is bound to the DNA before the cleavage reaction.

A modulated cutting pattern results; the absence of bands in the

sequencing pattern indicates the location of the ligand (89, 90).

This technique has also been used to measure the helical repeat of

B-conformation DNA (91).

3.5 Protein-DNA interactions

A gel electrophoresis DNA-binding assay has been developed for the

study of protein-DNA interactions (92-94). A major reduction in

mobility occurs when DNA fragments are complexed with protein molecules. The gel matrix appears to stabilize the protein-DNA complex,

122
although the mechanism is not understood. A "caging" effect has been

proposed: the gel matrix may form compartments that prevent the DNA

from diffusing away from the protein (93). Alternatively, the protein

and/or the DNA may interact with the gel matrix, or a discontinuity in

ligand concentration may exist, similar to that observed with spermine

(95).

3.6 Experimental procedures

The progressive increase in resistance which is sometimes observed in

polyacrylamide gel electrophoresis was studied by Spencer (95-97).

Changes in conductivity were attributed in part to the formation of a

zone of altered salt concentration at the interface between the gel

and the solution. A discontinuity in solvent concentration was observed, which migrated slowly into the gel. The effect was attributed

to a change in transport numbers of the ions on two sides of an interface (96). There may also be changes in pH within the zone of altered

concentration (97). Electrophoresis of tRNA in the presence of spermine

resulted in a progressive decrease in current when running at constant

voltage; the tRNA was contained in a sharp zone of precipitated material

within the gel, at a slowly moving discontinuity in salt concentration.

The region of the gel behind the moving boundary contained a much

lower concentration of spermine than the electrophoresis buffer.

Righetti, et al . (53) measured the pore size of polyacrylamide gels,

using polystyrene latex particles of known diameter. Highly diluted

polyacrylamide gels (2% acrylamide, 2.2% crosslinker) were found to be

123
gluey and did not admit the latex particles, suggesting that the

maximum pore size was smaller than 100 nm. Highly cross-linked gels

containing 30% acrylamide were prepared using several crosslinking

agents, including Bis. Below 25% Bis, no latex particles could enter

the gel. At 25% Bis, the gel structure became porus (100 nm average

diameter). The porosity increased rapidly up to 40% Bis, then increased at a slower rate up to 60% Bis. Gels containing more than 30%

Bis were hydrophobic and exuded water. Cross-linking agents with very

short chain lengths between the two terminal double bonds were needed

to obtain highly porous gels.

Highly porous gels containing as little as 2.5% acrylamide were prepared in a different manner by adding 0.6% linear polyacrylamide to

the gel matrix (98). The use of 0.4% dial lyl tartardiamide as a crosslinker gave clear gels without the loss of large pore size.

Acknowledgments

The work in the author's laboratory was supported by Grant GM-29690

from the National Institute of General Medical Sciences, which is

gratefully acknowledged.

124
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134
Table I

Mobil ity at Zero Gel Concentration

Type of M x 10 4 Author Reference DNA cm2 /Vsec.

Olivera, et al 3 T2, T4 fragments 1.51*

Ross & Scruggs 30 calf thymus 1.85*

Johnson & Grossman 31 PM2 1.4

Serwer & Allen 28 linearized pBR322 3.1

Edmonson & Gray 26 A-DNA restriction

fragments

1. 5-3.0

Stel lwagen 6 0.5-12 kbp fragments 2.0t

*free solution, 0.1 M NaCl

^mobilities extrapolated to zero electric field strength

135 -
Table

II

Birefringence

relaxation

time

of

DNA

fragments

in

free

solution

and

in

agarose

gels

of

different

concentrations

u

0) i- a> 4.

u o 3 co 3 u 3 u O E O 0) O

0) 0) +J *-> cO -*-> CO cn c c E E c o 3. e o rH E o C o a.

4-

C

u 4->

C

u «-> rHu 4-»

fT3

Oxd 3)XX

X c r>* <4-

C rH

X c c O o a> H CM o 1) o o 0) E

*H CM

X -H rH c

an

XX II o CM II a>

X II 8 o O o o o I-H rH 1 i-H r-H i-H co “J r-H i-H U

a>u a>

Uu o a> t- cO 3 u 3

CO 3 E O 0) o

a> 3L o 4-> co *-> cO 4-» rH C E c O X E c r-H E o E o

i.

C o -CC

u CM

C

u 4-> <3 -H u 4-> d 3) X X X X X) <*- C rH X 4— c < CO c*— C *H x o 01 X o <v E

P"- rH o 0)

HX c

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rH rH

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£-

C o -CCu 4-> i-H c u 4-> fO *H u 4-> d 3> X X CO x CO Vb. rH X c

«<

XX <*- c -H ao o 0)X o a> E

lD r-H o a» -H

X

C

CM

lO II II

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X _J

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CM II o d 1 Of X X CM —i X CM —J X p".

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u

u Sm

4-> co 3 U 3 U 3 3 V/) 3. o 3J o 1» o

<-> cO

H

w o 4. co E c 3. E c 3 E c X H— c o -C C o X C o JZ H o •*-> O u —> O u H Of | X X CO X) 3) X X X a; CM X w c o C r-*, c

4.

X »-H o O) •H CO o Of •H

X o 01 1 u_ CM

II II '

X II 1 O <1 *“ 1 d ^r O ^r 3 X CO X —1

*T

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a. csi CD X

xCMxroX

CM

at

vOD

a.xc

136
ABSTRACTS

THE FUNCTIONS OF A PROTEIN DATABASE DEVELOPED FROM CCMPUTER - ANALYZED

QUANTITATIVE TAO-DIMENS IONAL GELS. Stephen H. Blose, Laboratory for Cell

Biochemistry, Protein Databases, Inc., 405 Oakwood Road, Huntington Station,

New York 11746.

Protein databases are ind i spens i b 1 e sources of dynamic protein

information about living organisms. The information covers the spectrum of

regulatory behavior of all the detected proteins in a cell; it tells about

the proteins’ genetics - hence the organism’s genetics; it tells about the

proteins’ sub-cellular and tissue distribution; it tells which proteins are abnormal in various disease states; and protein databases will ultimately

tell us the function(s) of the proteins in a cell or tissue. A unique

property of protein databases is that they continue to evolve as more

information is learned by an investigator and as more information is

acquired from the scientific literature. The building blocks of protein

databases are derived from computer-analyzed quantitative two-dimensional

gel data obtained from many experiments, all referenced to a standard gel

pattern. The foundation of the database provides protein information on:

*PROTEIN IDENTIFICATION

*SUBCELLULAR LOCATION OF PROTEINS

* POST-TRANSLATIONAL MODIFICATIONS

* PARTIAL AMINO ACID (IMPOSITION

* RELATION OF PROTEIN PATTERNS TO CELL PHENOTYPE

*GENETIC EXPRESSION OF PROTEINS IN ORGANISMS

* PROTEIN PURIFICATION STRATEGIES

*ASSAY PROTEINS PRODUCED IN IN VITRO TRANSLATION SYSTBvIS

The ultimate utilization of protein databases is to provide investigators

with a better international medium to communicate results by providing

readily understood quantitative information. When key proteins are

identified, two-dimensional gels can rapidly purify the protein for antibody

production ot amino acid sequencing. This can be accomplished by using

a thick preparative two-dimensional gel. The amount of protein recovered

for each of the top 150-300 spots is in the pico-mole range. Protein

databases function in other scientific fields by providing information

on: protein purification, dynamic properties of protiens (factors that

influence protein synthesis and secondary modif icat ion, expression of mutant

proteins, rate of protein turn-over - the protein’s half-life), functional

protein complexes determined through chemical cross-linking and/or

inminoprecipitation, protein translocation though cell compartments, protein

binding properties (binding to DNA, lectins, and/or antibodies), cancer

studies, growth factor studies, genetic engineering and breeding, genetic

diseases, mutation and teratology studeis in animal and plant systems,

clinical trials and diagnostic tests, drug and tocicology reseach; studies

on single cells, cell lines, and cell senescence; quality control, and

studies in the neurosciences. Effective use of databases to solve problems

in each of these areas will be discussed.

137
GENETIC STUDY OF MARINE VERTEBRATE AND INVERTEBRATE POPULATIONS

USING ULTRA- THIN ISOELECTRIC FOCUSING TECHNIQUES

Yvon C . Chagnon

Bio-Conseil Inc.

2065 Boul . Cherest Ouest

Suite 119

Ste-Foy (Quebec) GIN 2G1

Two to eight different populations of two marine fish species and two marine

crustacean species were studied using double isoelectric focusing on short (5

cm) ultrathin polyacrylamide gels (0.2 mm). Six to 35 different enzymes were studied on 50 to 300 individuals within each species. Linear and non-linear pH

gradients were developed to obtain the best resolution of isoenzymes among

species by mixing ampholytes (Pharmacia) of different pH ranges. Techniques

were standardized for common reagents and showed species similarity. In

contrast, pH gradients and isoenzyme patterns varied to a great extent among

species, particularly for some enzymes.

138
DETECTION OF HEXOSAMINIDASE ISOZYMES BY SLAB GEL ISOELECTRIC FOCUSING (PAG-IEF)

Gail Chuck and George Hug, Children's Hospital Medical Center, Cincinnati, Ohio 45229

N-Acetyl-B -D-Hexosaminidase, a lysosomal enzyme, exists in two major forms: (1)

3-hexosaminidase A (Hex A), a heat-labile heteropolymer (a 6 ?

) cleaves G^ ganglioside,

G^ 7 and hexosamine oligosaccharides; (2) 3 -hexosaminidase B (Hex B), a heat-stabile

homopolymer (^ 3?) degrades G^, globoside and hexosamine oligosaccharides. Deficiency

of Hex A results in Tay-Sach's disease; deficiency of Hex A and Hex B produces SandhoffiJatkewitz disease. Clinical manifestations of these diseases range from early death to

late onset juvenile or adult forms and from normal clinical features to severe neurological

deterioration.

The enzyme is separated by IEF using an ultra thin PAG with a pH 4 to 9. A gel

consisting of 12% sucrose, 10% glycerol and 5% ampholyte with a T=5% and C=3% is cast on GelBond with a 2/100 inch spacer. The gel is prefocused for 1 hour with 5 mA constant

current at 10°C on the LKB ultraphor with 0.5 M H^PO^ and 0.5 M NaOH as electrolytes.

For optimal results, saturated LKB application pads of tissue culture homogenates, made

in 0.05 M citrate buffer pH 4.3 are applied at the midline. Electrophoresis begins with 5

watts, 5 mA and 500 volts and is increased periodically to 1500 volts for a total run of 4

to 5 hours. The active bands for Hex A and Hex B are visualized by: (1) an overlay of

freshly made substrate containing 10 mg napthol-AS-BI-N-Acetyl- 3-D glucosaminide

dissolved in 5 ml ethylene glycol monomethyl ether and mixed with 50 ml citrate buffer

pH 4.5 and 10 mg garnet GBC salt. With incubation at 37°C for 60 to 90 minutes, the

freed napthol couples with the fast garnet GBC producing a violet color. (2) an overlay of

0.1% 4 MUF N-acetyl-3-D-glucosamide in 0.1 M citrate buffer pH 4.5 incubated at 37°C

for 30 minutes. Fluorescence is viewed with long wave UV light.

Hex A and Hex B each exist in at least 6 distinct bands with pi range of 4.5 to 5.4

(Hex A) and 7.2 to 7.9 (Hex B). We determined these banding patterns in homogenates of

skin fibroblast cultures from patients with the various clinical forms of Tay-Sach,

Sandhoff-Jatkewitz, AB variant as well as from obligate carriers and normal controls. In addition, the isozyme patterns have been determined in homogenates of liver, placenta

and chorionic villi from normal individuals.

139
TITLE:

AUTHOR:

ABSTRACT:

NuFixTM Glyoxyl Agarose: A Versatile New Medium for Electrophoresis, Blotting and Chromatography

R. B. Cook, FMC BioProducts, 5 Maple Street, Rockland, Maine 048.

.

The wide spectrum of glyoxyl agarose properties will be

discussed

1) Its controllable reactivity toward 1° and 2° amines

2) Its native fixation capability

3) Its potential for affinity electrophoresis

4) Its exceptional pre- and postimmobilization stability and

5) Its ability to be cast in any format

Glyoxyl agarose/PAG composite gels permit in situ immobilizati r of polypeptides after electrophoretic separation.

The application of glyoxyl agarose to blotting and chromatograpwill also be noted.

140
THE USE OF BIOTINYLATED HUMAN DNA PROBES FOR DETECTING RFLP '

S

Human genomic DNA nick translated and labeled with bioten was used to

hybridize and detect restriction fragment length polymorphisms, (RFLP'S)

separated by electrophoresis and Southern blotted to nylon membranes. The

detection system was a streptavidin-biotin-alkaline phosphatase complex

which attached to the biotin ligand on the hybrids. The method was used to

detect RFLP'S with a variety of single copy probes and numerous restriction

endonucleases. The use of biotinylated probes for routine work was found

to be more economical, faster and safer than the use of radioactive probes

such as P32. RFLP'S could be detected with 2-5/^g of human DNA with an

electrophoresis, hybridization and staining procedure which could be

accomplished in less than 2 days.

Dale D. Dykes

Memorial Blood Center of Minneapolis

2304 Park Avenue South

Minneapolis, MN. 55404

141
T SC

1 _ •>. I '-YPSIC (PI) Ft

.0 , IC PC 'CCS IPO

v Yi-Cp ULTRA’i i 1 I iY hi

o c -StsoI , P.A., Bmcc Budovlo, Ph. . D . and 1 andal

1

-•ire1

' , Ph . D . , ?3I Academy, Quant ico, Virginia , 221?"

i .coel

-- ^ , v- on t,

• cj - O

r fnct ivo °coro.?ical method for the ultra thin la ver

metric focusing of alpha 1 — ant it ryps in (hi) was p°r-

• ’ us inn -r>v> a r ma 1 yt e 4.2— 4. 9. This procedure, with ar

r -

-i i—'~ '1 ] ?t 3 PC^ pf Q .

t- raso] vps t

1 - e ma mr c; 1

'

3 1

I ” 1

ir Ilf mi rut os on a 0.2 mm tl ich ooi ,

. r a crvl a m i d <•>

' r\ 'j ,o q

g sv ort °r irt nrwi cK h istanm^ of ”7.0 --m odvre t v at time to 7 5 minutes. 1 •<? S , ,

. 1 , ?,

copa ram r’i variant s arc r°?o] ’ ro 'n and at nasi 3 y

o

'1 hv Coor.a ssie Blue or silt'or stair inc. or cl i ra

i

3 '"t pq !- j n >, 1

3

o 3 r I - •' ’iptboc1 suitsbif3 o or tl e

^ pf )]'. p s j 00 ^^ficiort ja r 1 a nt vif 31m ^ evel 00

e

r

, ", 3. and --2 are separated in 3? mi nut os across

"i o 7-7.- 7 — -1 1 c * 1 -7 ‘O 37 ^ 0 o ^-r* ~\T ' 01 * 'S >• is r o

i

r molv

142
NEW DEVELOPMENT IN HARDWARE AND SOFTWARE FOR 2D GEL ANALYSIS

James I. Garrels, B. Robert Franza, Jr. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

We have development of protein databases for rat, mouse, and yeast cells

using a new hardware and software system for 2D gel analysis. The software,

based on our earlier QUEST system, has been converted and substantially

enhanced for use on a modern 32-bit workstation running under the UNIX

operating system. Features of the new system essential to database

development, and some examples from the database will be presented.

Protein databases must be contributed to and accessed by many users if they are to serve a useful function. The analysis of a major experiment (e.g.

10 gels of 1500 spots each) should not require more than overnight for the

automatic processing procedures and not more than a few hours of interactive

analysis by an operator. The Masscomp hardware, with its integral array and

graphics processor, clearly allows this level of throughput. Automatic processing

now includes automatic merging of all film exposures, accurate two-dimensional

gaussian fitting of all spots, creation of composite standard patterns, and rapid

matching and cross-matching.

New interactive features, such as rapid editing by spot quality, easy

comparison of the gel image and synthetic spot image at any magnification, and

the ability to refit spot clusters at any time allow user to verify and improve

the quality of the automatic processing within a short time.

Each database is begun by building a core of information that describes

the basic properties of the cells under study and their reproducibility under

standard experimental conditions. Statistical analysis of multiple gels, multiple

labelings, and multiple clones of cell lines are among the first experiments being

analyzed. Some of the statistical controls and some new findings from the

database will be presented.

143
AUTOMATED NUCLEIC ACID ELECTROPHORESIS AND HYBRIDIZATION ANALYSIS

D.M. Gersten, E.J. Zapolski, T.J. Golab, R.S. Ledley

Georgetown University Medical Center

Washington, D.C. 20007, USA

A computer controlled, fully automated system for Southern-type nucleic

acid hybridization analysis has been designed and constructed. DNA digested

with restriction endonucleases is loaded by the operator, into the sample

wells of the gel, which is contained in a nine-fingered plastic frame. The

operator then loads the labelled probe(s) into the hybridization chamber.

Instructions for all the steps in the automated process are specified by

the operator's answers to questions which appear on the computer screen at

the start of the experiment. Subsequent steps are performed automatically.

The system performs horizontal, submarine electrophoresis. An adjustable

endpoint detector terminates the electrophoresis. Automatic voltage/ temperature feedback control maintains maximum allowable voltage while keeping

temperature constant. Following electrophoresis a computer controlled

robot arm moves the gel frame from station to station. The system then fixes

the separated fragments to a solid phase matrix, denatures, neutralizes,

32 washes, dries and detects the P according to the specifications preprogrammed by the operator. The results, printed out by the computer, give a plot

of radioactivity versus distance from the origin for each of the nine

simultaneous hybridizations.

144
Electrophoretic Separation of High Density Lipoprotein Fractions.

Robert L. Gilman

Laboratory Service, V.A. Medical Center, Gainesville, Florida 32602

ABSTRACT

A clinical laboratory method is presented for use in the determination of

the relative quantities of the two major high density (alpha fraction)

lipoprotein fractions present in human serum. The method described was developed in this laboratory to help in the evaluation of coronary artery

disease risk factors for patients. Methods such as ultracentrifugation

can be used to determine HDL

2 /HDL

2 ratios, but they require special

equipment and great skill. This procedure involves removing the low

density lipoproteins from the serum sample, then fractionating the remaining

high density lipoproteins by electrophoresis on special gelatinized

cellulose acetate strips. Visualization is done by staining with a fat

stain, or by using a cholesterol reagent. The ratio is obtained by

scanning the wet strip on a densitometer.

145
PHYSICAL CHARACTERIZATION OF TURNIP CRINKLE AND

PELARGONIUM FLOWERBREAK VIRUSES BY AGAROSE GEL

ELECTROPHORESIS: APPLICABILITY OF POLYSTYRENE SIZE

STANDARDS AND OF MOVING BOUNDARY ELECTROPHORESIS BUFFER

SYSTEMS. E. Gombocz, D. Tietz, S. Hurtt and A. Chrambach , NIH,

Bethesda MD Z089Z and " USDA, Beltsville MD Z0705.

Turnip Crinkle and Pelargonium Flowerbreak Viruses were physically

characterized by quantitative agarose gel electrophoresis at pH 6.5,

0.03 M ionic strength, 2 °C, 10 to 50 mM CHAPS, using N-&-

hydroxyethy lmorpholinium-ACES buffer either continuously in all phases

(2 V/cm) or as the gel buffer within a discontinuous buffer system (1.2

2 mA/cm ). Agarose (Iso-Gel, LKB) at concentrations ranging from 0.1 to

2.3% was applied in standard thermostated gel tube apparatus. Viruses

were reacted with ethidium bromide prior to electrophoresis and

detected by the bound fluorescence. Polystyrene size standards

(Polybead Microspheres, Polysciences, Warrington PA) ranging in nominal

radius from 28.5 to 75 nm were visible zones.

Stacking of both the viruses and the polystyrene standards in 0.4%

agarose required a) trailing and leading ion net mobilities relative to

Na of 0.059 and 0.273 respectively; b) CHAPS concentrations of 50 mM in

2 all phases; c) current densities not exceding 1.2 mA/cm .

Non-linear Ferguson plots ( log(mobility) or log(R^) vs. agarose

concentration) were obtained for viruses and standards. The slopes of

the lines connecting points on the curves with the curve intercepts on the mobility axis, KD

' , were determined. Coefficients obtained by

curve-fitting for the standards were applied to the appropriate

functions to compute particle radii of the viruses on the basis of

K ' values.

K

146
Immobilized pH gradients for one and two-dimensional

separations. Applications.

Angelika Gorg, Wilhelm Postel, Siegfried Gunther and

Johann Weser. Techniscne Universitat Munchen.

D-6050 Freising-Weinens tephan (F.R.G.).

A high degree of resolution and reproducibility is achieved

with isoelectric focusing in narrow (< 0.2 pH units /cm) and

ultra-narrow (0.05 - 0.01 pH units /cm) immobilized pH

gradients (IPG). Human plasma proteins (e.g. Gc, Tf, Pi)

analyzed for genetic variability are distinctly resolved

and located for accurate subtyping (1,2,3). By using narrow

IPG in the first dimension closely related proteins, which

tend to be clustered into small areas of the 2D map, are

better resolved (4)

.

Proteins from yeast and legumes are

focused in broad IPG (pH 4-10) in the presence or absence of

urea, detergents or carrier ampholytes and applied to the

second dimension. The resulting 2D maps differ essentially

reflecting the findings obtained with IEF in three different

systems (IEF, IPG, IPG-CA) . In comparitive experiments, two

identical IEF strips were applied to a horizontal and a vertical oDJ pore gradient gel. The 2D maps show no significant

differences with respect to resolution, spot size and distrioution, altnougn the horizontal system is easier to perform

than the vertical system. The equilibrated IEF gel strip on

plastic support is simply loaded on the surface of the

norizontal SDS pore gradient gel (500 ym thick on Gel Bond

PAG film). Wo agarose overlay or embedding gels are needed (5)

(1) H. Cleve, W. Patutschnick, W. Postel, J. Weser and A. Gorg. Electrophoresis _3, 342-345 (1982)

(2) A. Gorg, J. Weser, R. Westermeier, W. Postel, S. Weidinger,

W. Patutschnick and H. Cleve. Hum. Genet. 64_, 222-226 (1983)

(3) A. Gorg, W. Postel, J. Weser, W. Patutschnick and H. Cleve. Am. J. Hum.

Gen. 37_, 922-930 (1985)

(4) A. Gorg, W. Postel, J. Weser and R. Westermeier. In Electrophoresis

'83 (Hirai, H. ed) , de Gruyter, Berlin 1984, pp. 525-532

(5) A. Gorg, W. Postel, S. Gunther and J. Weser. Electrophoresis 6_, 599-604 (1985)

147
A NEW PROGNOSTIC INDICATOR IN ACUTE LYMPHOBLASTIC LEUKEMIA DETECTED

BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS (2-D PAGE).

Samir M. Hanash, Leslie J. Baler, and Rork Kuick. Department

of Pediatrics, Univeristy of Michigan Medical School, Ann Arbor,

Michigan

.

In previous studies we have utilized 2-D PAGE to detect polypeptide

markers in acute lymphoblastic leukemia (ALL). Sixteen polypeptides

were detected that, as a group, distinguished between CALLA positive

non-T, non-B; CALLA negative non-T, non-B; B cell and T-Cell

ALL. Over the past four years 72 patients with ALL have been

studied. One polypeptide, L3 (M W 29,000) was observed in all

53 CALLA positive and nine CALLA negative non-T , non-B ALL patients,

but was not detected in 6 T-cell and 4 B-cell ALL cases nor in

15 patients with AML. In contrast to other markers, substantial

variability in the intensity of L3 was observed between patients.

The variable intensity of L3 led us to search for clinical or

pathological correlates. A strong correlation was observed between

the intensity of L3 and clinical outcome. Of 18 patients with

a faint L3, 10 have relapsed; ;whereas 3/19 with a moderately

intense and 1/15 with a heavy L3 spot have relapsed. Of the patients

who have been followed in our study for two years or longer, 9/10

with a faint L3 spot have relapsed; whereas none of 10 patients

with an intense spot have relapsed. There was no correlation

between the intensity of L3 and other established prognostic indicators.

Therefore, the detailed analysis of the polypeptide constituents

of leukemic cells, as made possible by 2-D PAGE, has revealed

a previously undetected polypeptide, the deficiency of which is

associated with an unfavorable outcome in non-T, non-B ALL.

148
Mechanistic Studies of Silver Staining

Diane K. Hancock

Center for Analytical Chemistry

National Bureau of Standards

Gaithersburg, MD 20899

Much controversy exists as to the mechanism of silver stains and to the

importance of several variables. We are attempting to probe the mechanism of

the stain or stains and to find answers to questions such as: Does the Ag+

bind specifically to the protein and if so to what groups in the protein? Is

the binding ratio characteristic of the protein? Does the binding step

determine the overall sensitivity of the stain, and if so how can we adjust

conditions (e.g. pH, ionic strength, temperature) to maximize the binding? Why

is the Ag+ reduced mainly in the vicinity of the protein and how can we

minimize non-specific (e.g. background) reduction?

We have carried out preliminary silver-protein binding studies using a variety

of measurement techniques. Bovine serum albumin was titrated with 10 mol/L

AgNC>3 using a Ag ion specific electrode to monitor Ag concentrations. Neutron

activation analysis has been used to map Ag concentrations in polyacrylamide

slab gels that were stained with either a Merril-type silver stain or with an

ammoniacal silver stain. The feasibility of using *~ (^Ag NMR to study silverprotein binding sites has been investigated. Results of these studies will be

presented.

149
Studies on Catechol-O-Methyltransferase in Rat Brain

Using Two-Dimensional Gel Electrophoresis

William E. Heydorn, G. Joseph Creed, Cyrus R. Creveling and David M. Jacobowitz

Laboratory of Clinical Science, NIMH and Laboratory Bioorganic Chemistry

NIADDK, NIH, Bethesda, MD. 20892

The presence of catecho 1-0-methyl transferase (EC 2. 1.1. 6) in the rat brain was studied using a combination of two-dimensional gel electrophoresis, protein

blotting and a specific antiserum. Two major immunoreactive proteins were identified - one with a molecular weight of 23 kDa and an isoelectric point of

5.2, the other of molecular weight 25 kDa and isoelectric point of 5.1. In addition, multiple lower molecular weight immunoreactive proteins, possibly

corresponding to breakdown products of the enzyme, were also detected. The

23 kDa form of catechol-O-methyltransferase, which is probably the soluble

form of the enzyme, is a major protein visible on silver-stained

two-dimensional gels of rat brain. In contrast, the other proteins recognized

by the antiserum were not detected by the silver stain.

These results demonstrate, using two-dimensional gel electrophoresis, that at

least two distinct forms of catechol-O-methyltransferase are present in rat

brain. In addition, since one of these proteins is stained by silver, these

results also serve to identify another protein visible on two-dimensional

e lectrophoretograms of rat brain.

150
IMMOBILINE ELECTROPHORESIS: pi VALUES FOR PROTEINS OF LDH-4 AND -5 FROM

CONTROL AND NITROSOMETHYLUREA-TRANSFORMED NEOPLASTIC CELL LINES.

Ann E. Kaplan and Glenn N. Gray, Laboratory of Experimental Pathology,

National Cancer Institute, FCRF Bldg. 560, Frederick, MD 21701-1013

Agarose and PAG electrophoresis of LDH-4 and LDH-5 from a control cell line

established from rat hepatocytes, TRL, and its nitrosomethylurea-transformed

neoplastic cell line, NMU-3, shows that the proportions of the two isozymes

differ in the two cell lines. In TRL cells, LDH-4 is three to four times

more active than LDH-5, whereas in NMU-3 cells the reverse is observed.

Following PAG electrophoresis, three faint protein bands are detected with

the silver stain method of Wray and Wray (Anal. Biochem. 118 , 197, 1981).

Two proteins are parallel to the two LDH isozymes but a third is not and

does not react as LDH. First attempts to identify the pi values of the two

isozymes were carried out with the isoelectric focussing (IEF) method.

LDH-4 separated into 5-6 proteins which migrated below pH 7; LDH-5 migrated

to the alkaline region of the gel, to at least pH 8.8, but could not be

better-defined in this system. With the application of Immobiline electrophoresis (IE) the results to date are as follows: Eight proteins are

distributed between LDH-4 and -5. LDH-4 is characterized by six proteins

with pi values ranging between pH 5.3 and 6.8. LDH-5 is characterized by

two proteins with pi values above pH 9.0. The results with IE confirm the

wide pH difference between the pi values of the proteins associated with

each of the two LDH isozymes. The two proteins identified thus far with

LDH-5 appear to have pi values above pH 9. These values are among the

highest reported thus far, only cytochrome c being above pH 10.

151
PARAMETRIC VERSUS NON-PARAMETRIC GEL IMAGE ANALYSIS

* Reinhold C. Mann, Betty K. Mansfield, James K. Selkirk

Biology Division, Oak Ridge National Laboratory, P.0. Box Y

Oak Ridge, Tennessee 37831

*National Toxicology Program, NIEHS, P.0. Box 12233,

Research Triangle Park, North Carolina 27709

ABSTRACT

Two different sets of methods tor spot detection and quantification in digital

images generated by two-dimensional gel electrophoresis have been described in

the literature: (1) parametric methods that operate on the basis of a Gaussian

spot model, and (2) non-pa rametric methods that do not rely on any assumptions

concerning the shape of spots. There are no data available that would allow to

judge the relative advantages and disadvantages of either approach to gel image

analysis.

Therefore, we performed a set of experiments involving computer simulations in

order to compare the accuracy of quantification of two typical methods, subsequently referred to as A and B from classes (1) and (2) respectively, implemented on a general purpose image analysis system. The simulated images

consisted of a number of Gaussian spots with differing degrees of overlap to

which random noise with Gaussian distribution density was superimposed at different signal to noise ratios. Only isolated spots were included in the analysis.

Preliminary results indicate that the differences in relative errors and variability of the estimates between methods A and B depend on spot intensity in case

of a non-linear OD-dprn relationship. The coefficient of variation (cv) of

method B estimates tends to be lower than the cv obtained with method A. This was particularly well noticed for OD ranges for which the slope of the OD-dpm

curve was steepest. This is due to the fact that errors committed during an imperfect Gaussian fitting process in the OD domain are amplified in the dpm domain through the non-linear OD-dpm conversion. In addition to considerations

regarding the amount of data reduction achieved by methods A or B, computational

efforts involved, ability to regenerate synthetic gel images tor visual inspection, etc., these results are of great importance in selecting algorithms tor an optimum gel image analysis system.

This research was sponsored by the Office of Health and Environmental Research,

U.S. Department of Energy, under contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.

By acceptance of this article, the publisher or recipient acknowledges

the U.S. Government's right to

retain a nonexclusive, royalty f rn

license in and to any copyright

covering the article

152
2 , 5-DI PHENYLOXAZOLE (PPO) IN DIMETHYL SULFOXIDE (DMSO) REGENERATION

FOR RE-USE IN FLUOROGRAPHY

* Betty K. Mansfield, Reinhold C. Mann, James K. Selkirk

Biology Division, Oak Ridge National Laboratory, P.O. Box Y

Oak Ridge, Tennessee 37831

*National Toxicology Program, NIEHS, P.O. Box 12233,

Research Triangle Park, North Carolina 27709

ABSTRACT

At present, impregnation of the scintillator PPO dissolved in DMSO into

two-dimensional electrophoresis gels for fluorography, as described by Bonner

and Laskey in 1974, remains one of the most sensitive and widely used methods

14

for detecting and quantifying radioactivity in tritium- and C-labeled proteins. The relatively high cost of this scintillator often necessitates the

reuse of PPO from partially depleted solutions. In an attempt to recover PPO

from the DMSO solvent for further use, researchers have precipitated PPO with

water, filtered, and dried it. We have developed a simple fluorescence assay

for measuring the amount of PPO remaining in a partially depleted solution of

PPO in DMSO. A calculated amount of PPO can then be added back to the partially

depleted solution to restore the initial PPO concentration. Thus, PPO solutions

can be regenerated and used many times before being precipitated due to the accumulation of water.

This research was sponsored by the Office of Health and Environmental Research,

U.S. Department of Energy, under contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.

By acceptance of this article, the publisher or recipient acknowledges

the U.S. Government's right to retain a nonexclusive, royalty-free

license in and to any copyright

covering the article 153
ANALYSIS OF MICRO-VARIATIONS IN THE RATE OF SYNTHESIS OF INDIVIDUAL

POLYPEPTIDES ISOLATED FROM CULTURES OF THE RAT HEPATOMA CELL LINE H4-

II-E AS MEASURED BY QUANTITATIVE TWO-DIMENSIONAL GEL ELECTROPHORESIS,

by Mark J. Miller and Snorri S. Thorgeirsson, Laboratory of Experimental

Carcinogenesis, National Cancer Institute, Bethesda, MD. 20892.

Living cells maintain themselves in a delicate metabolic balance,

adjusting to changes in their external and internal environments through

a wide range of biological processes. One of these processes is gene

expression, which manifests itself as protein synthesis. Twodimensional polyacrylamide gel electrophoresis has been used in numerous studies to detect modulation in the rate of protein synthesis as cells

progress through the cell cycle, are subjected to heat shock, or undergo

neoplastic transformation. Many of these changes are quantitative,

reflecting a change in the rate of synthesis rather than a turning on or off of synthesis. To date, we know of no study aimed at determining the

baseline level of variability in steady state cell cultures.

Using a computerized analysis system capable of detecting changes

in the rate of protein synthesis of as little 30%, we have examined the

rate of synthesis of over 900 polypeptides in two independent subclones

of the rat hepatoma cell line, H4-II-E (Reuber Cells). Cells from both

subclones were grown in the same media lots, and all experiments were

done on log phase cells at approximately the same cell concentration.

Only 7 proteins were detected that were Qualitatively different between

the two subclones; i.e. a spot was consistently present in one subclone,

but undetectable in the other. Some of these spots may be modification

products of one another. On the other hand, 9% of the spots showed

statistically significant quantitative differences when compared, in

triplicate, between the two subclones. Duplicate extractions of the same culture indicate these differences are not artifacts of sample

preparation or electrophoresis. Multiple experiments indicated that,

although there was always a significant degree of heterogeneity in the

quantitative expression of proteins between the two subclones, these

differences involved different sets of proteins from experiment to

experiment. A time course experiment, in which samples were taken and

analyzed every week for 5 weeks indicated that the expression of certain

polypeptides can vary significantly during the life of the culture.

These experiments indicate that the differences observed represent a dynamic within the cell culture and not, generally, a difference between

the two subclones. The nature and cause of this variation is under

investigation.

154
^-Hemoglobin Polymorphism in White-Tailed Deer: Suitability and

Application to Wildlife Law Enforcement.

Rodger Morrison and Morris G. Brown

The ^-hemoglobin types of over 250 Virginia white-tailed

deer (Odocilus virginianus) have been determined by cellulose

acetate electrophoresis. Nine different genotypes have been

observed in the sample from the deer population of the Skyline

Management Area, in Jackson County, Alabama. The homozygous

type III was found to be the most common (86.6%). Gene

frequencies for the five Hb^ alleles are as follows: II (0.028),

III (0.921), IV (0.014), V (0.028), VII (0.008). All genotypes

other than the homozygous type III were confirmed by isoelectric

focusing in thin acrylamide gels, pH 5-7.

In forensics^ species identification of deer blood has been

the main objective, particularly in areas where closely related

species are found. This represents a step towards intraspecies

individualization in white-tailed deer. ^-hemoglobin appears to

be a stable marker. Both cellulose acetate electrophoresis and

isoelectric focusing were found to be reliable procedures for the

determination of ^-hemoglobin types. The method has been used in

a wildlife law enforcement case involving a deer killed in the

Skyline Management Area.

Criminalist and Forensic Lab Analyst respectively

Alabama Department of Forensic Sciences

716 Arcadia Circle

Huntsville, Alabama 35801

155
PREPARATION OF URINE AND SERUM SAMPLES WITH

CENTRIFUGAL MICROCONCENTRATORS AND TOTAL PROTEIN

ANALYSIS BY ULTRAVIOLET-DIFFERENCE SPECTROSCOPY

FOR TWO-DIMENSIONAL ELECTROPHORETIC ANALYSIS

James E. Myrick, Mary K. Robinson, Ivey Lois Hubert, Carol J. Bell,

and Samuel P. Caudill, DEHLS, CEH, Centers for Disease Control, Public

Health Service, U.S. Department of Health and Human Services, Atlanta.

CA 30333.

Two-dimensional electrophoresis (2DE) requires concentrated and

desalted urine samples or diluted and desalted serum. We have

developed procedures for the ultrafiltration and di af

i

1 trat ion of

preserved (sodium azide, sodium carbonate, and aprotinin) urine and serum with centrifugal microconcentrators fitted with 10 , 000-dal ton

cutoff filters. Parallel processing with a 24-position rotor allows

up to 20 mL of each urine sample to be ul traf i ltered in 12-14

centrifugation steps, which achieves approximately a 200-fold

concentration, and then diafiltered with water in 2 steps. This

processing is completed in about 2.5 days. The filtration rate for

urine averaged 1.3 mL/cent r i f ugat i on (6,000 x g for 30 min) and only

sightly decreased with each centrifugation. Retention of

I- beta -2-microglobulin (M =11,800 daltons) on the filters wa3

virtually 100%, and < 10 , 000-dal ton proteins were absent on calibrated

and silver-stained 2DE gels of urine or serum, confirming the sharp

M cutoff. After the di af i Itration step, the retentates of 24 human

u'rine samples were analyzed for total protein bv

ultraviolet - difference spectroscopy, Coomassie dye binding,

bicinchoninic acid (BCA), and Lowry methods. The UV-d i f f er ence method

compared most favorably with the Lowry method, was the easiest to

implement, and required no additional reagents other than protein

calibrators and water diluent. Urinary 2DE protein patterns from

normal samples prepared as above showed at least one major group of

spots, which is absent from samples prepared with gel exclusion

methods and which is removed by cellulose nitrate filtration. The

microconcentrator method is suitable for large studies in which

reproducibility over long periods is important.

156
TITLE: Effects of Borate on Agarose Gel Structure

AUTHORS

ABSTRACT:

Stephen Peats, Sam Nochumson, Francis H. Kirkpatrick

FMC BioProducts, 5 Maple Street, Rockland, Maine 04841

Borate buffers change the sieving properties of agarose

gels when compared to non-borate buffers. In both agarose

(SeaKem® LE) and low-melt agarose (SeaPlaque®, NuSieve©) , substitution of borate for acetate increases the apparent

sieving of DNA fragments (72bp to 3000bp, ca. 40,000 kD to

2MD) . The sieving increase due to borate is most pronounced

if the borate is present during casting of the gel.

Borate also alters agarose viscosity, gel strength, and

gelling and melting temperatures. Using an analysis

similar to that of Serwer et al (Electrophoresis 4, 232-236,

1983) , it appears that borate may bind to agarose in such a way as to reduce the packing density of helices during gel

formation.

157
Results of an Interlaboratory Round Robin using Isoelectric Focusing to

Assess Reproducibility of Rehydratable Gels

K. L. Richie and D. J. Reeder

National Bureau of Standards

A- 361 Chemistry Bldg

Gaithersburg, MD 20899

During the past year, the National Bureau of Standards conducted an

interlaboratory comparison study to test the effectiveness of rehydratable

gels in obtaining consistent patterns with standard protein preparations.

Each of five laboratories was provided with a package containing standard

protein samples, rehydration media with instructions, techniques for silver

staining and ampholines. Laboratories were allowed to run gels by their

existing methods or whatever method they felt would give the best results.

In comparing laboratory results it was noted that there were

distinguishable variations in the protein band patterns of the same protein

due to several factors affecting the electrophoretic run. Factors

identified include: 1) dependence on electrode separation distance,

voltage and time of run; 2) nature of the electrolyte solutions;

3) quantity of sample applied and method of application; 4) type of

apparatus used; and 5) relative humidity at the time of the run.

Of the above, it appears that the type of apparatus used was a major factor

in producing differences. The second most significant difference appeared

to be the electrode separation distance.

A second round robin needs to be run taking into account the above

factors. In addition, the general running conditions will be specified, if

possible, and anolyte and catholyte solutions will be provided along with

suggestions for sample application.

158
ELEVATED LEVELS OF p21 ras DETECTED IN BLAST CELLS FROM LEUKEMIC

PATIENTS.

Atul Sahai and Samir M. Hanash. Department of Pediatrics, University

of Michigan Medical School, Ann Arbor, Michigan.

Activation of the cellular oncogene ras has been implicated

in many types of human malignancies including hematopoietic tumors.

However, most studies of ras oncogene expression have examined

either the ability of DNA derived from tumor cells to transfect

NIH 3T3 cells or the level of mRNA in the cells, and few have

examined the protein products of the celllular oncogenes. In

the present study: 1) We have analyzed the polypeptide patterns

of lymphocytes obtained from the peripheral blood of healthy individuals

and of blasts from leukemic patients using two-dimensional polyacrylamide

gel electrophoresis (2-D PAGE). Protein patterns were visualized

by silver staining and f luorography . 2) We report the expression

of the p21 ras product in blast cells from various leukemic patients.

Lymphocytes from healthy donors and from leukemic patients were

simultaneously labelled with -^S-methionine (200 uci/ml) for overnight

and cell lysates were immunoprecipitated using monoclonal antibody

Ab-1 (Oncogene Sciences, New York) that detects C-Ha-ras, C-Ki-ras

and N-ras forms of the protein. Untransformed NIH-3T3 cells and

3T3 cells transformed by viral Harvey ras were used as negative

and positive controls, respectively. 2-D PAGE of immunoprecipitates

detected elevated levels of p21 ras in some of the leukemic patients.

The amount of p21 ras observed was below the detection threshold

of silver staining techinique as well as f luorography . Under

the conditions used in our study while it apperas that the p21

polypeptide product of ras oncogene is not detectable in leukemic

cells by either silver staining or f luorography , several other

polypeptide alterations were observed. The significance of these

alterations remain to be determined.

159
INTERMEDIATE RANGE MOLECULAR WEIGHT MARKERS FOR SDS GELS

David Sammons and Robin Humphreys, Center for Separation Science,

University of Arizona, Tucson, AZ 85721

Molecular weight markers for SDS and 2-D electrophoretic gels are essential for intra and inter-laboratory comparisons of protein

patterns. The molecular weight markers used presently with

Coomassie blue stains are not suited for ultrasensitive silver

stains because of microheterogeneity from contaminating proteins,

proteases, and oxidation of sulfhydryls . The test criteria applied

in selecting ideal markers are band thickness, pattern stability,

purity, color, linearity, and reproducibility. Four markers,

having apparent molecular weights of 81,000, 40,500, 29,000 and

17,500 daltons are recommended as intermediate range molecular

weight markers for SDS gels.

160
The Effect of Calcium Ion Concentration on the Electrophoretic Mobility of Two

Cultured Human Embryonic Kidney Cell Populations

By Burton E. Sarnoff, Debra Berretta and Paul Todd. Bioprocessing and

Pharmaceutical Research Center, 3401 Market St. /Suite 220, Philadelphia, PA 19104

A very low ionic strength buffer is used for the Continuous Flow Electrophoretic Separator (CFES) operated by NASA and McDonnell-Douglas Astronautics Co.

The absence of sodium and low levels of other ions in this buffer provides an

opportunity to study the effects of particular ions on the Electrophoretic

Mobility (EPM) of cells. Human Embryonic Kidney (HEK) cells were used in this

experiment because they are used in an ongoing NASA project to electrophoretically purify urokinase producing HEK cells and because they are electrokinetically heterogeneous. A Pen Kern System 3000 Automated Electrokinetic Analyser

was used to measure the EPM of two cultures of HEK cells derived from different

sources. Calcium ion concentration was varied from the standard CFES

concentration of 0.0274 mK to 27.4 m>l. As expected on the basis of published

results and theoretical considerations, increased calcium ion concentration led

to decreased EPM, with overall population heterogeneity remaining about the same.

The lowest mobility subpopulations of cells had near zero or positive mobility

at the highest calcium ion concentrations.

This work was supported by the U.S. National Aeronautics and Space

Administration.

161
Advantages of Focusing Car bamy 1 at i on

Trains in a Flat-bed System

Cheri Seitz

Pharmacia Biotechnology Group

Pharmacia, Inc.

Car bamyl at i on trains are usually visualized in the

second dimension of two-dimensional el ec tr ophor esi s systems.

This can be a lengthy and labor-intensive process. Our need

to compare the carbamy 1 at i on patterns of many different

proteins requires a quicker method, one which would allow

simultaneous running and viewing of several samples.

Our method is a pol yacry 1 ami de flat-bed isoelectric

focusing gel of the same recipe as one might use in the

first dimension of a 2-D system. This gives several

advantages over running rods:

- separate lanes on one flat gel are easier to

compare than separate rods which may vary in length

- the sample application site is not limited to

the anode or cathode

- extended pH gradient due to less cathodic drift

- flat surface allows use of a mi croel ectrode for

apparent pH determination

- easier to handle than rods

This system allows a rapid comparison of separations

occurring during the isoelectric focusing dimension of the

2-D system.

This poster will outline our flat-bed isoelectric

focusing method and show the results obtained.

162
TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELETROPHORESIS OF LYMPHOCYTE PROTEINS

WITH IMMOBILIZED pH GRADIENTS IN THE FIRST DIMENSION. John R. Strahler,

Samir M. Hanash, Luke Somerlot. Department of Pediatrics, University

of Michigan Medical School, Ann Arbor, Michigan and Angelika Gorg,

Lehrstuhl fur Allegemeine Levensmittel Technologic der Technischen

Universitat Munchen, Germany.

We have investigated the use of isoelectric focusing in immobilized

pH gradient (IPG) gels as the first dimension in two-dimensional polyacrylamide

gel electrophoresis of lymphocyte proteins. IPG gels were compared

in the range between pH4 and 10. Washed, dried and rehydrated IPG

gels were run in the presence of 8M urea, NP-40, dithiothreitol , and

the presence or absence of free carrier ampholytes. Lymphocyte proteins,

solubilized with 9M urea, 2% NP-40 and carrier ampholytes, were applied

to individual IPG gel strips. Gels were focused for 1 hr (1 w constant

power) until the sample had entered the gel and then for 5 hr at 5

w constant power (3,000 v maximum). The strips were then equilibrated

for varying times at pH 6.8 or 8.6 with urea, SDS and mercaptoethanol

,

and applied to the second dimension SDS polyacrylamide gradient gel.

Polypeptides were visualized by silver staining. The majority of polypeptides

visualized have acidic pi's (pH range 4-6). Of particular interest

however were the polypeptides seen in the alkaline region. Polypeptides

with pi's in the range 7 to 9 do not focus well or are not seen in

our 2-D gels using conventional isoelectric focusing with carrier ampholytes.

The inclusion of the non-ionic detergent NP-40 in the IPG gel disturbed

the focusing in the alkaline range but the effect was minimized by

using shorter focusing times. Carrier ampholytes in the IPG gel increased

the net transport of protein into the gel, nevertheless there was significant

precipitation of sample at the site of application. The use of IPG

gels in the pH range 7 to 10 and containing carrier ampholytes, urea

and NP-40 will make it possible to analyze the basic proteins of lymphocytes

and other cell types.

163
PHYSICAL CHARACTERIZATION OF PARTICLES AND GEL FIBERS ON

THE BASIS OF NON-LINEAR FERGUSON PLOTS IN AGAROSE GEL

ELECTROPHORESIS. D. Tietz , E. Gombocz and A. Chrambach , NIH } Bethesda

MD 20892.

The non-linear Ferguson plots observed in the quantitative agarose

gel electrophoresis of viruses (Serwer et al., Gombocz et al.), other

subcellular particles (Gottlieb et al.) and polystyrene microspheres

(Gombocz et al.) call for an evaluation of particle radii and gel fiber

properties on the basis of a retardation coefficient variable with gel

concentration (Tietz et al . ) . Mobility data of proteins, viruses and

polystyrene particles were analyzed by computer simulations, using

program MLAB (DCRT, NIH) . The applied mathematical model uses functions

derived from the Ogston theory. Parameters which originally were

constants are replaced by variables dependent on gel concentration and

particle size. Correspondingly, the retardation coefficient K

K (which is constant for each particle when its Ferguson plot is linear)

is replaced by a variable K

' defined for each gel concentration by the

slope of the line connecting a point on the curve with the curve intercept on the mobility axis.

Computer modeling 1) accounts for the apparent paradox of different

fiber radii derived from the agarose gel electrophoresis of proteins and

viruses, by demonstrating a continuous variation of effective agarose

fiber radius on gel concentration; 2) demonstrates a dependence of

effective agarose fiber radius on particle size; 3) indicates

compressibility of polystyrene microspheres at high agarose

concentrations; 4) allows one to compute particle radii of unknowns in

the range of 15 to 80 nm, using commercial polystyrene microspheres as size standards.

164
PREPARATIVE AND ANALYTICAL IMMOBILINE ELECTROFOCUSING OF CLASS III HEPATIC

ALCOHOL DEHYDROGENASE. K.H. Valkonen and D. Goldman (Unit of Genetic Studies,

Laboratory of Clinical Studies, NIAAA, Bethesda, Maryland 20892).

We have resolved human and rat hepatic Class III alcohol dehydrogenases

(ADH) by analytical and preparative immobiline electrofocusing. Separations

were carried out in narrow (pH 5.3-6. 9) immobilized gradients. Both in

human and in rat there were three bands which stained with cinnamol but

not with ethanol and which were insensitive to the inhibitory effect of

pyrazole. The i sol electric points of the Class III ADH bands, determined

in analytical gels by using commercial isoelectric markers, were 6.3, 5.9

and 5.6. The three Class III ADH bands from human liver showed the same

isoelectric points in preparative immobiline gradients as in analytical

gels. The enzyme electroeluted from each Class III ADH band was active

with the long chain alcohol cinnamol (7.5 mM), was not saturated even by 2 M

ethanol, and was not inhibited by 12 mM 4-methyl pyrazole. Polyacrylamide

gel electrophoresis in the presence of sodium dodecyl sulfate revealed

that the eluted Class III ADH bands consisted primarily of a single 43 kd

molecular species corresponding to Class III ADH, and lesser amounts of a

65 kd contaminant. Immobiline isoel ectrofocusing thus provides a sensitive

analytic method for the detection of charge variants and a rapid preparative

separation method for Class III ADH.

165
Sequential Analysis of Chemically Induced Hepatoma Development

Using Two-Dimensional Electrophoresis (2D-PAGE)

Peter J. Wirth and Snorri S. Thorgeirsson

Laboratory of Experimental Carcinogenesis

National Cancer Institute, Bethesda, MD 20892

It is becoming increasingly apparent that cancer development in

many tissues or organs is a stepwise process involving altered cell

populations at many steps. The liver offers a very attractive model for

the study of carcinogenesis since the process of hepatocarcinogenesis

appears to follow a "programmed" series of distinctive cellular and tissue changes which ultimately result in the formation of hepatocellular

carcinomas. It seems likely, however, that the acquisition of the neoplastic phenotype would bring about both qualitative and quantitative

changes in cellular functions that are distinctly different from those

observed under normal and/or preneoplastic conditions. Utilizing a

number of experimental carcinogenesis protocols our laboratory has

undertaken the sequential analysis of polypeptide changes during hepatocarcinogenesis in the rat using 2D-PAGE. Analysis of 1000-1200 silver

stained cytosolic and membrane-associated polypeptides under both

equilibrium and nonequilibrium (NEPHGEL) conditions revealed four qualitative and numerous quantitative polypeptide differences between normal

liver and preneoplastic (PN) and neoplastic (N) tissues. One cytosolic

(pi 6.8/57 kDa) and three membrane-associated polypeptides (6.25/41

,

6.75/24, and 6.05/21) were expressed in both PN and N tissues but not in

normal liver. In addition roughly 4-10% of the polypeptides were undergoing quantitative changes of at least four-fold during these stages of

hepatocarcinogenesis. These data suggest that gene expression is not

extensively altered during early stages of heptocarcinogenesis.

166
FOUNDATION EXPERIMENTS FOR BUILDING A PROTEIN DATABASE. Virginia P. Wray,

Protein Databases , Inc, 405 Oakwood Road, Huntington Station, New York

11746.

Building the foundation of a protein database that organize the

protein information can be initiated by conducting several initial

experiments and examining the results on computer-analyzed two-dimensional

gels. The initial experiments include:

1. Determine the optimal conditions to radiolabel the

system’s proteins steady-state vdth various

aminoacids (i.e., [ S] -methionine , [ ^H] - 1 euc i ne , ['"TC] -pro line)

.

2. Map all the protein ’’players” in the system on several

combinations of isoelectric focusing gels and

second-dimension acrylamide gels.

3. Quantitate the amino acid composition of all the

proteins in the system to establish specific

’’signatures” for each protein.

4. Determine secondary post-translational modifications of

the proteins as part of their signature and

regulatory behavior (phosphorylation,

glycosalat ion, methylation, fatty acid acylation,

etc . . . ) where appropriate.

5. Pulse-chase studies to determine the turnover (halflife) of all resolved proteins.

6. Other suggested experiments:

A. Identify key known proteins and enzymes by

mixing in purified proteins and by

immunopr ec ipi ta t i on to establish key

landmarks

.

B. Compare and Quantitate:

>Prote in changes after various

experimental manipulations such as:

drug d o s e - r e s p on s e , growth,

development, etc.

>Protein differences produced by genetic

manipulations (map phenotypic

changes)

.

>Proteins derived from cell

fractionation studies to determine

the protein’s subcellular location.

>Proteins obtained from different tissues

of an organism to establish tissue

specific prote in marker s

.

The assembled data constitutes a protein fingerprint for an experimental

cell or tissue model. This type of database continues to evolve as more

information becomes available from experimentation and the scientific

literature. Protein databases can be utilized in many areas of cell biology

and medicine to accelerate accumulation of new research data.

167
MECHANISM OF NICKEL AND SILVER VISUALIZATION

of PROTEIN ELECTRO PH ORE GRAMS

Dr. Joseph Yudelson

Research Laboratories

Eastman Kodak Co., Rochester, New York 14650

The so-called metal stains have found increasing use in protein

electrophoresis, particularly where improved sensitivity is desired.

Such visualization systems are examples of what is called physical

development in the photographic field, and electroless plating in

the metal plating industry. The determination of the sensitivity

and amplification factors of these systems is based on well-known

principles of heterogeneous catalysis.

A description of the chemistry, nucleation, and amplification

processes involved in nickel and silver visualization systems will

be presented, together with calculations describing their ultimate

sensitivity

.

168
COMPARISON OF 2-DIMENSIONAL GEL ELECTROPHORETIC SEPARATIONS USING

SOFT LASER SCANNING DENSITOMETRY WITH COMPUTER COLOR GRAPHICS

Rashid A. Zeineh, Biomed Instruments, Inc., 1020 S. Raymond Ave.

#B, Fullerton, California 92631

Two autoradiograms of two-dimensional slab gels (5"x5") showing

electrophoretic separations of normal and cancerous mouse skin

fibroblasts were individually scanned using a computer-aided soft

laser scanning densitometer. The contour of the abnormal state

(red color) was overlaid electronically on that of the normal

(green color) sample on the screen for direct comparison. In this

way the user was enabled to detect the appearance (red) or disappearance (green) of protein spots in the test sample at a glance.

The quantitative differences were shown in the computer printout

in the form of tabulated numerical values such as, spot number,

X-Y position, peak height and amount of protein. In the event the

images could not be superimposed properly due to gel distortion

the user was enabled to manipulated the data in the test sample

while the control data remained on the screen fixed. Flexible

superimposition was accomplished using the following functions:

(1) slide: right, left, up or down; (2) expand - compress: in

the X or Y axes and (3) rotate: clockwise or counterclockwise.

In general, the system was found easy to use and it facilitated

quantitative and qualitative comparison of complex protein spot

patterns revealed on two-dimensional gel electrophoregrams in a

time-saving manner.

169
MULTILANE STRIP-CHART PRINTING PROGRAM FOR DNA SEQUENCE READING

Rashid A. Zeineh, Biomed Instruments, Inc., 1020 S. Raymond Ave.

#B, Fullerton, California 92631

A computer system has been developed which allows superimposition of 4 lanes (A, G, C & T bases) on chart paper, in different

colors, for determining the position of bases shown on X-ray film.

The system when used in accord with a high resolution- high sensistivity soft laser scanner offers the following distinct advantages: (1) Ensures accurate DNA sequence reading, since determining the position of a base among different lanes, becomes an

easier and more precise process when bases are identified as

"peaks" in densitometric tracings rather than as "bands" in electrophoretic separations on autoradiograms. (2) Electronic expansion of the four-lane graphics (A, G, C & T ) provides clear and

easy reading of bases and especially when graph paper is used

for computer printout. The vertical lines on the paper serve as

reference points that help to determine the relative base positions more accurately. In addition to this, DNA sequence reading

is further facilitated by printing of each of the four tracings

in different color. This feature provides distinct identification of the individual lanes, facilitating thus reading of base

sequence.

The system described for DNA sequence reading is a powerful analytical tool which eliminates ambiguities as well as tedious and

time-consuming work inherent to manual methods.

170
AUTHOR INDEX

Leslie J. Baier 148

Carol J. Bell 156

Debra Berretta 161

Stephen H. Blose 137

Morris G. Brown 155

Bruce Budowle 1 Bruce Budowle 142

Samuel P. Caudill .... 156

Yvon C. Chagnon 138

A. Chrambach 146

A. Chrambach 164

Gail Chuck 139

Richard B. Cook 140

G. Joseph Creed ..... 150

Cyrus R. Creveling .... 150

Dale Dykes 141

John Fawcett ........ 13

B. Robert Franza, Jr. . . 143

Anne M. Gambel 142

James I. Garrels 143

Douglas M. Gersten .... 144

Robert L. Gilman 145

T. J. Golab 144

D. Goldman 165

E. Gombocz 146

E. Gombocz 164

Angelika Gorg 147

Angelika Gorg 163

Glenn N. Gray ...... 151

Siegfried Gunther .... 147

Samir M. Hanash 148

Samir M. Hanash 159

Samir M. Hanash 163

Diane K. Hancock 149

William E. Heydorn .... 150

Ivey Lois Hubert 156

George Hug 139

Robin Humphreys 160

S . Hurtt 146

David M. Jacobowitz . . . 150

Ann E. Kaplan 151

Francis H. Kirkpatrick . . 157

Rork Kuick 148

R.S. Ledley 144

Reinhold C. Mann 152

Re inhold C. Mann 153

Betty K. Mansfield .... 152

Betty K. Mansfield .... 153

Carl R. Merril 36

Carl R. Merril 66

Mark Miller 154

Rodger Morrison 155

Randall S . Murch 142

James E. Myrick 156

Sam Nochumson 157

Susan C. Olson 66

Stephen Peats 157

Wilhelm Postel 147

Dennis J. Reeder 158

Kristy L. Richie 158

Mary K. Robinson 156

Atul Sahai 159

David Sammons 160

Burton E. Sarnoff .... 161

Cheri Seitz 162

James K. Selkirk 152

James K. Selkirk 153

Philip Serwer 88

Luke Somerlott 163

Nancy Stellwagen 104

John R. Strahler 163

Snorri S. Thorgeirsson . . 154

Snorri S. Thorgeirsson . . 166

Dietmar Tietz 146

Dietmar Tietz 164

Paul Todd 161

Kaija H. Valkonen .... 165

Johann Weser 147

Peter J. Wirth 166

Virginia P. Wray 167

Joseph Yudelson 168

E.J. Zapolski 144

Mike Zeineh 169

Mike Zeineh 170

Jack Zeineh 169

Jack Zeineh 170

Rashid A. Zeineh 169

Rashid A. Zeineh 170
.
SUBJECT INDEX

Agarose Gel Electrophoresis 88, 164 Moving Boundary Electrophoresis

Buffer Systems 146

Alcohol Dehydrogenase 165

Non-Linear Ferguson Plots 164

Calcium Ion Concentration 161

Nucleic Acid Electrophoresis 144

Carbamylation Trains 162

Peptides 66

,

154

Catechol -0 -Methyl transferase 150

Pheno typing 142

Characterization of Particles

and Gel Fibers 164 Polystyrene Size Standards 146

DNA 104 Prognostic Indicator 148

DNA Sequence Reading 170 Protein Database 137, 167

DNA Probes 141 Rehydratable Gels 158

Fluorography 153 Scanning Densitometry 169

Gel Analysis 143 Silver Staining 36, 149

Glyoxyl Agarose 140 Slab Gel Isoelectric Focusing 139

Hepatoma Development 166 Total Protein Analysis 156

High Density Two-Dimensional Gel Electrophoresis

Lipoprotein Fractions 145 148, 150, 154, 156, 163, 166, 169

Hybridization Analysis 144 Ultra-Thin Layer Isoelectric Focusing

1, 138, 142

Image Analysis 152

Viruses and Viral Components 88, 146

Immobilized pH Gradients

13, 147, 151, 163, 165

Interlaboratory Round Robin 158

Isoelectric Focusing 158

Isozymes 139

Law Enforcement 155

Levels of p21 ras 159

Lymphocyte Proteins 163

Mechanism of Nickel

and Silver Staining 149, 168

Molecular Weight Markers 160

NBS-I14A 1REV. 2-80

U.S. DEPT. OF COMM.

BIBLIOGRAPHIC DATA

SHEET (See instructions)

1. PUBLICATION OR

REPORT NO.

NBSIR/86—3345

2. Performing Organ. Report No. 3. Publication Date

MARCH 1986

4. TITLE AND SUBTITLE

Proceedings of the 1986 Meeting of the Americas Branch of the Electrophoresis

Society

5. AUTHOR(S)

Editor: Dennis J. Reeder

6. PERFORMING ORGANIZATION (If joint or other than NBS, see instructions)

national bureau of standards

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

7. Contract/Grant No.

8 . Type of Report & Period Covered

9.

SPONSORING ORGANIZATION NAME AND COMPLETE ADDRESS (Street, City. State, ZIP)

10.

SUPPLEMENTARY NOTES

| | Document describes a computer program; SF-185, FlPS Software Summary, is attached.

11.

ABSTRACT (A 200-word or less factual summary of most si gnificant information. If document includes a significant

bi bliography or literature survey, mention it here)

This publication consists of submitted plenary papers and abstracts for the

1986 Meeting of the Americas Branch of the Electrophoresis Society. The plenary

papers are in-depth reviews and comprise 80% of the document.

12. KEY WORDS (Six to twelve entries; alphabetical order; capitalize only proper names; and separate key words by semicolon s) Electrophoresis; isoelectric focusing; nucleic acid electrophoresis;

peptide electrophoresis; staining methodology; immobilized pH gradients

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