NOAA Technical Report NMFS 61 December 1987

Manual for Starch Gel

Electrophoresis: A Method for

the Detection of Genetic Variation

Paul B. Aebersold, Gary A. Winans, David J. Teel, George B. Milner,

and Fred M. Utter

U.S. DEPARTMENT OF COMMERCE

National Oceanic and Atmospheric Administration

National Marine Fisheries Service
NOAA TECHNICAL REPORT NMFS

The major responsibilities of the National Marine Fisheries Service (NMFS) are to monitor and assess the abundance and geographic distribution of fishery resources, to

understand and predict fluctuations in the quantity and distribution of these resources, and to establish levels for their optimum use. NMFS is also charged with the development

and implementation of policies for managing national fishing grounds, development and enforcement of domestic fisheries regulations, surveillance of foreign fishing off United

States coastal waters, and the develop,ment and enforcement of international fishery agreements and policies. NMFS also assists the fishing industry through marketing service

and economic analysis programs, and mortgage insurance and vessel construction subsidies. It collects, analyzes, and publishes statistics on various phases of the industry.

The NOAA Technical Report NMFS series was established in 1983 to replace two subcategories of the Technical Reports series: "Special Scientific Report-Fisheries" and

"Circular." The series contains the following types of reports: Scientific investigations that document long-term continuing programs of NMFS; intensive scientific reports on

studies of restricted scope; papers on applied fishery problems; technical reports of general interest intended to aid conservation and management; reports that review in considerable detail and at a high technical level certain broad areas of research; and technical papers originating in economics studies and from management investigations..Since

this is a formal series, all submitted papers receive peer review and those accepted receive professional editing before publication.

Copies of NOAA Technical Reports NMFS are available free in limited numbers to governmental agencies, both Federal and State. They are also available in exchange for

other scientific and technical publications in the marine sciences. Individual copies may be obtained from: U.S. Department of Commerce, National Technical Information Service,

5285 Port Royal Road, Springfield, VA 22161. Although the contents have not been copyrighted and may be reprinted entirely, reference to source is appreciated.

13. Guidelines for reducing porpoise mortality in tuna purse seining, by James M.

Coe, David B. Holts, and Richard W. Butler. September 1984, 16 p.

14. Synopsis of biological data on shortnose sturgeon, Acipenser breviroslrum

LeSueur 1818, by Michael J. Dadswell, Bruce D. Taubert, Thomas S. Squiers, Donald

Marchelle, and Jack Buckley. October 1984, 45 p.

15. Chaetognatha of the Caribbean sea and adjacent areas, by Harding B. Michel.

October 1984, 33 p.

16. Proceedings of the Ninth and Tenth U.S.-Japan Meetings on Aquaculture, by

Carl J. Sindermann (editor). November 1984, 92 p.

17. Identification and estimation of size from the beaks of 18 species of cephalopods

from the Pacific Ocean, by Gary A. Wolff. November 1984,50 p.

18. A temporal and spatial study of invertebrate communities associated with hardbottom habitats in the South Atlantic Bight, by E. L. Wenner, P. Hinde, D. M. KnOll,

and R. F. Van Dolah. November 1984, 104 p.

19. Synopsis of biological data on spottail pinfish, Diplodus holbrooki (Pisces:

Sparidae), by George H. Darcy. January 1985, II p.

20. Ichthyoplankton of the Continental Shelf near Kodiak Island, Alaska, by Arthur

W. Kendall, Jr., and Jean R. Dunn. January 1985,89 p.

21. Annotated bibliography on hypoxia and its effects on marine life, with emphasis

on the Gulf of Mexico, by Maurice L. Renaud. February 1985, 9 p.

22. Congrid eels of the eastern Pacific and key to their Leptocephali, by Solomon

N. Raju. February 1985, 19 p.

23. Synopsis of biological data on the pinfish, Lagodon rhomboides (pisces:Sparidae),

by George H. Darcy. February 1985, 32 p.

24. Temperature conditions in the cold pool 1977-81: A comparison between southern

New England and New York transects, by Steven K. Cook. February 1985,22 p.

25. Parasitology and pathology of marine organisms of the world ocean, by William

J. Hargis, Jr. (editor). March 1985, 135 p.

26. Synopsis of biological data on the sand perch, Dipleclrum formosum (Pisces:

Serranidae), by George H. Darcy. March 1985, 21 p.

27. Proceedings of the Eleventh U.S.-Japan Meeting on Aquaculture, Salmon

Enhancement, Tokyo, Japan, October 19-20, 1982, by Carl J. Sindermann (editor).

March 1985, 102 p.

28. Review of geographical stocks uf tropical dolphins (Slenella spp. and Delphinus

delphis) in the eastern Pacific, by William F. Perrin, Michael D. Scott, G. Jay Walker,

and Virginia L. Casso March 1985, 28 p.

29. Prevalence, intensity, longevity, and persistence of Anisakis sp. larvae and Lacislorhynchus lelluis metacestodes in San Francisco striped bass, by Mike Moser, Judy

A. Sakanari, Carol A. Reilly, and Jeannelle Whipple. April 1985,4 p.

30. Synopsis of biological data on the pink shrimp, Pandalus borealis Krllyer, 1838,

by Sandra E. Shumway, Herbert C. Perkins. Daniel F. Schick, and AJden P. Stickney.

May 1985, 57 p.

31. Shark catches from selected fisheries offthe U.S. east coast, by Emory D. Anderson, John G. Casey, John J. Hoey, and W. N. WitzelI. July 1985, 22 p.

32. Nutrient Distributions for Georges Bank and adjacent waters in 1979, by A. F.

J. Draxler, A. Malle, R. Waldhauer, and J. E. O'Reilly. July 1985,34 p.

33. Marine flora and fauna of the Northeastern United States. Echinodermata:

Echinoidea, by D. Keith Serafy and F. Julian Fell. September 1985,27 p.

34. Additions to a revision of the shark genus Carcharhinus: Synonymy of Aprionodon and Hypoprion, and description of a new species of Carcharhinus (Carcharhinidae),

by J. A. F. Garrick. November 1985, 26 p.

35. Synoptic review of the literature on the Southern oyster drill Thais haemasloma

j1oridana, by Philip A. Butler. November 1985, 9 p.

36. An egg production method for estimating spawning biomass of pelagic fish:

Application to the northern anchovy, Engraulis mordax, by Reuben Lasker (editor).

December 1985, 99 p.

37. A histopathologic evaluation of gross lesions excised from commercially important North Atlantic marine fishes, by Robert A. Murchelano, Linda Despres-Patanjo,

and John Ziskowski. March 1986, 14 p.

38. Fishery atlas of the northwestern Hawaiian Islands, by Richard N. Uchida and

James H. Uchiyama (editors). September 1986, 142 p.

39. Survey of fish protective facilities at water withdrawal sites on the Snake and

Columbia Rivers, by George A. Swan, Tommy G. Withrow, and Donn L. Park. April

1986, 34 p.

40. Potential impact of ocean thermal energy conversion (OTEC) on fisheries, by

Edward P. Myers, Donald E. Hoss, Walter M. Matsumoto, David S. Peters, Michael

P. Seki, Richard N. Uchida, John D. Ditmars, and Robert A. Paddock. June 1986,

33 p.

41. A stationary visual census technique for quantitatively assessing community structure of coral reef fishes, by James A. Bohnsack and Scott P. Bannerol. July 1986, 15 p.

42. Effects of temperature on the biology of the northern shrimp, Pandalus borealis,

in the Gulf of Maine, by Spencer Apollonio, David K. Stevenson, and Earl E. Dunton,

Jr. September 1986, 22 p.

43. Environment and resources of seamounts in the North Pacific, by Richard N.

Uchida, Sigeiti Hayasi, and George W. Boehlert (editors). September 1986, 105 p.

44. Synopsis of biological data on the porgies, Calamus arclifrons and C. proridens

(Pisces: Sparidae), by George H. Darcy. September 1986, 19 p.

45. Meristic variation in Sebasles (Scorpaenidae), with an analysis of character association and bilateral pallern and their significance in species separation, by Lo-chai Chen.

September 1986, 17 p.

46. Distribution and relative abundance of pelagic nonsalmonid nekton off Oregon

and Washington 1979-84, by Richard D. Brodeur and William G. Pearcy. December

1986, 85 p.
NOAA Technical Report NMFS 61

Manual for Starch Gel

Electrophoresis:

A Method for the Detection

of Genetic Variation

Paul B. Aebersold, Gary A. Winans, David J. Teel,

George B. Milner, and Fred M. Utter

December 1987

u.s. DEPARTMENT OF COMMERCE

C. William Verity, Jr., Secretary

National Oceanic and Atmospheric Administration

Anthony J. Calia, Administrator

National Marine Fisheries Service

William E. Evans, Assistant Administrator for Fisheries
The National Marine Fisheries Service (NMFS) does not approve, recommend

or endorse any proprietary product or proprietary material mentioned in this

publication. No reference shall be made to NMFS, or to this publication furnished

by NMFS, in any advertising or sales promotion which would indicate or imply

that NMFS approves, recommends or endorses any proprietary product or proprietary material mentioned herein, or which has as its purpose an intent to cause

directly or indirectly the advertised product to be used or purchased because of

this NMFS publication.

11
CONTENTS

Introduction

Collection and storage of specimens

Preparation of tissues 3

Preparation of a starch gel 4

Application of enzyme extracts to a gel 6

Setting up a gel for electrophoresis 7

Slicing a gel 8

Staining a gel 9

Acknowledgments 10

Citations 10

Appendices

I Staining recipes 11

2 Electrophoresis buffers 20

iii

Manual for Starch Gel

Electrophoresis:

a Method for the Detection

of Genetic Variation

PAUL B. AEBERSOLD

GARY A. WINANS

DAVID J. TEEL

GEORGE B. MILNER

FRED M. UTTER

Coastal Zone and Estuarine Studies Division

Northwest and Alaska Fisheries Center

National Marine Fisheries Service, NOAA

2725 Montlake Boulevard East

Seattle. Washington 98112

ABSTRACT

The procedure to conduct horizontal starch gel electrophoresis on enzymes is

described in detail. Areas covered are (I) collection and storage ofspecimens, (2)

preparation of tissues, (3) preparation of a starch gel, (4) application of enzyme

extracts to a gel, (5) setting up a gel for electrophoresis, (6) slicing a gel, and (7)

staining a gel. Recipes are also included for 47 enzyme stains and 3 selected gel

buffers.

Introduction _

Electrophoresis is the most useful technique yet devised for studying genetic variability within and among populations of plants and

animals (Avise 1974; Ayala 1976; Hartl 1980). In fact, over 1,200

species have been studied electrophoretically in basic and applied

research (see references in Nevo et al. 1984). Since 1966, geneticists at the Northwest and Alaska Fisheries Center, National

Marine Fisheries Service, NOAA, Seattle, Washington, have been

using starch gel electrophoresis of proteins (or, more specifically,

enzymes) to investigate population genetics of numerous aquatic

organisms, particularly fishes.

Electrophoresis is the separation of a mixture of electrically

charged molecules in an electric field. When a molecular mixture

is placed in an electric field, the molecules with a net electrical

charge will be attracted to the opposite side of the electric field

(positive molecules to the negative side and negative molecules to

the positive side) at a rate dependent on the amount of their charge

(and to some extent on their size). Therefore, all like-charged

molecules will migrate a characteristic distance through the electric

field in a given time. When a mixture is applied in the field, the

result is an isolation of each molecular charge-type at varying

distances from their point of application.

The purpose of this report is to thoroughly describe procedures

currently used in our laboratory for horizontal starch gel electrophoresis. It is intended as a complement to other descriptions

that may be outdated or abbreviated (Smith 1968; Brewer and Sing

1970; Utter et al. 1974; Harris and Hopkinson 1976; Siciliano and

Shaw 1976; May et al. 1979; Shaklee and Keenan 1986). The

procedures we describe have been adopted more from personal

experience and convenience than from necessity. Thus, other laboratories may effectively employ procedures that differ from ours.

The general areas and procedures we cover, however, are common

to all users. Despite specific procedural differences, they yield the

same result. The areas we discuss in detail are (1) collection and

storage of specimens, (2) preparation of tissues, (3) preparation of

a starch gel, (4) application of enzyme extracts to a gel, (5) setting

up a gel for electrophoresis, (6) slicing a gel, and (7) staining a gel

(Fig. I). Recipes for 47 enzyme stains (Appendix 1) and 3 gel

buffers (Appendix 2) are included. We believe that this information will be generally useful as a working manual because electrophoretic investigations of all classes of organisms are related by

similar biological constraints (e.g., heat-labile enzymes) and mechanical processes involved in separating proteins in a gel matrix.

In addition, the thoroughness with which we have covered these

procedures will be particularly useful for people attempting starch

gel electrophoresis for the first time.

Collection and storage of specimens _

Procedures for the collection and storage of specimens for electrophoresis are extremely important because of the lability of enzymes in vitro. Proper cryogenic storage will preserve enzyme

activity and minimize breakdown. Conversely, improper collection and preservation commonly result in a reduction or loss of

enzyme activity or in uninterpretable electrophoretic patterns due

to enzyme breakdown. Thus, the ultimate success of an electrophoretic project rests initially on the proper collection and

preservation ofspecimens. Although the procedures described here

are for fish specimens, the processes are generally applicable to

other organisms.
1 Collection and storage of specimens

2 Preparation of tissues

4 Application of enzyme extracts to a gel

•

tfr----__

/ .

5 Setting up a gel for electrophoresIs .

6 Slicing a gel

7 Staining a gel

Figure 1

Diagram of the seven steps involved in the electrophoretic method.

2
When fish are sacrificed for analysis, enzyme degradation begins almost immediately. The rate of degradation is enzymespecific. Packing the specimens in ice will slow the degradation

process only slightly. Freezing the samples at normal freezer temperatures (-lO°C to -20°C) will reduce degradation considerably.

However, even at these temperatures, if electrophoresis is not

conducted within a few weeks, enzyme degradation will become

apparent. At the temperature of dry ice (-76°C) or colder, the

stability of the enzymes is greatly improved. Enzyme activity in

tissues immediately frozen with dry ice and stored for up to 6 mo

at -85°C in an ultracold freezer is comparable to enzyme activity

in tissues from freshly sacrificed specimens. In fact, we have

achieved satisfactory results with specimens collected on dry ice

and stored at -85°C for greater than 2 yr. Thus, for the best

electrophoretic results, we recommend that specimens or tissues of

specimens be (l) frozen with dry ice immediately after collection,

(2) transported with dry ice to an electrophoresis laboratory, and

(3) stored at -85°C until used.

The quantity of dry ice needed while collecting in the field

depends upon many factors. These include the quality and size of

the cooler in which the samples are held, volume of specimens

being collected, number of times the cooler will be opened prior to

returning to the laboratory, length of time spent in the field and in

transport, and ambient air temperature. For example, we would use

a minimum of 2.3 kg (5 Ib) of dry ice in a 7.6-L (8 qt) cooler to

collect 100 juvenile fish on a 24°C (80°F) day, with a total time of

8 h spent going to the sample location, collecting the specimens,

and returning to the laboratory. To collect tissues from 100 adults

under the same circumstances would require a 38-L (40 qt) cooler

and a minimum of 9.0 kg (20 Ib) of dry ice. Reducing the air space

in the cooler makes dry ice more effective and longer lasting. Also,

when sufficient dry ice is available, it is placed above and below

the specimens; otherwise, the dry ice is placed above the specimens in the cooler. Specimens are kept in a cooler with sufficient

quantities of dry ice until they can be stored in a - 85°C freezer.

Whole specimens or tissue samples are prepared for cryogenic

storage in a way that minimizes the amount of time necessary to

prepare them for electrophoresis. For example, it is undesirable to

freeze fish in a ball with water. Fish prepared in this fashion require

considerable thawing time to separate them for dissection, and,

therefore, they are more prone to enzyme degradation.

We recommend that whole fish be prepared for freezing in the

following manner which allows for easy separation prior to dissection. Fish with a fork length of 20 cm or less (five or more fish per

pound) are collected and frozen whole. Of these fish, those that are

6-20 cm long (between 5 and 160 fish per pound) are rolled up dry

in bundles of 10 to 20 specimens using sheets of polyethylene film

or "plastic wrap." To do this, specimens are briefly blotted to

remove excess moisture. Two or three specimens are spread out

near one end of the plastic wrap, which is then folded over the

specimens. Then, several more specimens are laid down, rolled

into the bundle, and so on. This method keeps the fish separated

by plastic film and facilitates separating them later for dissection.

Fish smaller than 6 cm fork length (160 or more fish per pound) are

loosely placed in a single layer into a sealable or "zip-lock" bag

(lOx 10 em) without water.

For specimens greater than 20 em fork length (five or fewer fish

per pound), we usually dissect out the desired tissues at the time

of collection and freeze the set of tissues from each specimen in

one zip-lock bag (lOx 10 cm). We routinely analyze enzymes from

four sources: skeletal muscle, heart, liver, and retinal tissue and

vitreous fluid from the eye. For the first three tissues, a cube of

3

tissue measuring approximately 3 cm3 or weighing 20 g is taken

from the fish when possible. For the retinal tissue and vitreous

fluid, both eyes are removed and stored whole. Care is taken to

avoid prolonged exposure of the tissue to direct sunlight. Each bag

of specimens from a location is labeled with information regarding

species name, date of collection, sample location, and number of

specimens. Labels are written in large bold print on water-resistant

paper and inserted in each bag so that they are visible from the

outside of the plastic bag. Alternatively, a tag on a piece of wire

can be used for this information as well as to secure the bag.

Preparation of tissues _

Once specimens or tissue samples have been obtained and stored

properly, an enzyme extract of each tissue is prepared for each

specimen. To do this, specimens are removed from the freezer and

allowed to thaw for about 10 to 20 min, just until the tissues can

be cut. When preparing subsamples of extracted tissues from adult

fish, approximately 0.75 g or 7 mm3 are cut from each tissue. To

subsample frozen eyes, one-fourth to one-third of the eyeball is cut

away and the frozen vitreous fluid with the surrounding black

retinal tissue is collected. Each of these subsamples is placed into

a culture tube (I2x75 mm, disposable) containing a tissue dilution

buffer. This is done for each tissue of each fish, being careful to

ensure the subsamples are not contaminated with other tissues. Our

tissue dilution buffer (abbreviated PIP buffer)I consists of PIPES

[Piperazine-N,N'-bis(2-ethanesulfonic acid)], Triton X-IOO, and

pyridoxal-5-phosphate. The volume of buffer added to each tube

depends upon the number of gels required for each specimen as

well as the amount and type of tissue being diluted. Usually about

0.2 to 0.4 ml of PIP buffer is added per culture tube. Muscle

tissues are usually given 0.05 mL more dilution buffer per sample

than liver or heart tissues because muscle tissues tend to absorb the

buffer. Because of their initial liquid state, eye fluid samples from

salmonids arc usually given one-half the volume of the liver or

heart tissues. We have observed that the vitreous fluid from marine

fish, however, is much more viscous than that of salmonids and

requires more dilution, 0.1 mL less than liver or heart tissues.

We routinely prepare tissues from 5 to 10 frozen specimens at

a time. The culture tubes are kept in a tube rack which is in an

icewater bath to keep the samples as cold as possible during the

preparation process. When the group of 5 to 10 specimens is

completed, the tubes are transferred into other tube racks, one

appropriately labeled rack for each tissue, and put into a freezer.

A specimen number, corresponding with the subsample position in

the culture tube rack, is added to each specimen bag in the event

resampling might be necessary. Upon completion of subsampling,

the racks of culture tubes are individually enclosed in plastic bags,

secured with rubber bands, and moved to a -85°C freezer until the

tissues are analyzed. We do not routinely homogenize tissues prior

to electrophoresis. Instead, we rely on a freeze-thaw process during subsample preparation and application to the starch gel for cell

disruption and enzyme extraction (see Aebersold et al. 1986).

However, tissues which will be used fresh or without extended

freezing should be homogenized in some fashion.

'The PTP buffer consists of 0.05M PIPES. 0.05% Triton X-lOO, and 0.2 mM

pyridoxal-5-phosphate adjusted to a final pH of 6.8 with 1.0 M NaOH. PIPES will

not go into solution at the initial acidic pH until an approximate pH of 5.5 is

approached with the NaOH. After that, the Triton X-IOO and pyridoxal-5-phosphate

are added before the final pH adjustment is made (Aebersold et al. 1986).
The preparation of whole juvenile fish for electrophoresis is

similar to the preparation of extracted adult tissues. How::ver,

dissection and extraction of the tissues involve a more delicate

procedure. Quite often the entire liver, heart, and one or both

eyeballs are taken from juvenile fish. In such cases, the heart

should be chopped up with a scalpel (to open the tough membrane

surrounding the heart) before insertion into the culture tube, and

eyeballs should be broken open in the tube by smashing them with

a glass rod. Also, the amount of dilution buffer should be reduced

in proportion to the reduction in tissue volume.

Preparation of a starch gel _

In the electrophoretic procedures described here, enzymes are separated in an electric field relative to their electrical charge to reveal

possible genetic differences. The medium we use for the electric

field to separate the enzymes is a starch gel. A gel is made in a

Plexiglas/glass mold into which hot starch solution is poured and

allowed to gel upon cooling.

The gel mold consists of a glass plate on the bottom and four

Plexiglas strips clamped around the edges (Fig. 2). We use laminated glass to eliminate stress fracturing that may occur when the

hot gel solution is poured into the mold. Dimensions of glass plates

and Plexiglas strips are given in Table I. The thickness of the strips

can be varied to make thicker or thinner gels as needed. The strips

are held in position on the glass plate with paper clamps. The mold

is elevated above the lab counter to leave clearance for the clamps

and accelerate cooling. To save time and counter space, a number

of gel molds can be set up together by butting the gel molds up to

one another before clamping, as in Figure 2. Gel molds should be

set up on a level surface. The type of buffer used to make the gel

is written with wax pencil on one of the short plastic strips for each

mold.

The gels are made by mixing a heated buffer with hydrolyzed

potato starch (Sigma Chemical Co., St. Louis, MO). In this procedure, the first step is to weigh the starch and put into a wide-mouth

Erlenmeyer flask. For one to two gels, a I-L flask works well; up

to four gels can be poured from a 2-L flask. We usually make a

12% starch gel, with a 6-mm thick gel mold holding 300 mL of

starch solution. Therefore, to make one gel, 36 g of starch are

weighed and added to the flask.

The buffer used to prepare the gel can vary considerably in

composition, concentration, and pH, each having an effect on the

mobility, resolution, and activity of each enzyme. Many buffer

recipes are available for these purposes. A number of these recipes

can be found in the references previously cited in the Introduction.

We use three buffers (or slight variations of these) in resolving a

majority of the enzymes in our studies. These buffers are given in

Appendix 2.

Each buffer is made as a concentrated stock solution and diluted

as stated to make the gel. After dilution, one-third of the volume

(100 mL of the 300 mL used for one gel) goes in the flask with the

starch, and the flask is immediately swirled to fully suspend all the

starch. (If a wet flask is used for the starch, the buffer should be

added immediately after adding the starch to reduce the chance of

creating lumps.) The remainder of the buffer is put into an

appropriate-sized volumetric or narrow-mouth Erlenmeyer

flask.

The flask containing only the buffer is heated. When the buffer

begins to boil, the flask is first swirled to settle the boiling action,

which could cause the buffer to splatter out when dispensing. Then

the buffer is rapidly poured into the flask with the starch-buffer

mixture, with simultaneous swirling of both flasks. It is important

to swirl the flask with the starch just prior to addition of the hot

buffer to ensure that the starch is fully suspended to achieve a

smooth lump-free gel. At this point, the starch mixture is a thick

paste. Additional heating is needed to raise the temperature of the

solution to the boiling point. This is necessary to achieve the

proper viscosity for aspiration and pouring. Proper viscosity is

indicated when the size of the bubbles changes from large to small

and the bubbles rise up to the surface of the starch solution. The

Figure 2

Gel molds. Four Plexiglas strips (1) are held with clamps (2) around each glass plate (3) to form a gel mold.

Hot liquid starch (4) is then poured into the mold as illustrated. The glass plates are elevated off the counter

on polystyrene cups (5) to leave room for the clamps.

4
Table 1

Dimensions of various items used in the electrophoretic process.

Length Width Thickness

Items --------._- (mm) ------------

For gel molds

glass plate 267 165 5.7

shon Plexiglas strip 165 19 6*

long Plexiglas strip 229 19 6*

For slicing gels

glass plate 280 152 5

Plexiglas strip 305 19 1*

Blue lee package 254 165 30

Cooling plate 267 165 25

Sponge cloths 235 127

*Strips twice as thick can be used depending upon the number of gel

slabs requir~d.

Figure 3

Degasing a solution of hot starch. The large flask (1) contains the hot solution. Water coming out ofthe spigot

(2) creates a suction in the aspirator (3) which draws out the air in the large flask as well as gas trapped in

the starch solution. The smaller flask (4) serves as the trap for catching any starch solution that may

inadvertently be drawn from the large flask.

mixture is swirled frequently during this time to reduce the chance

of burning the starch.

After heating, the starch solution is degased using a water aspirator connected to a trap (Fig. 3). The trap collects any starch

solution that might be sucked out of the flask. A hose with a rubber

stopper connects the trap to the flask. A hole in the stopper allows

control of the vacuum by pressing or raising a finger over the hole,

as shown in Figure 3. To begin aspiration, the rubber stopper is

placed into the flask opening. The spigot to the aspirator is turned

on full-force, and the hole in the stopper is covered with one finger.

The small bubbles should rise out of the starch solution, if sufficiently heated, as the solution begins to boil vigorously under this

vacuum. If the starch solution bubbles up close to the stopper,

some of the vacuum is released by slightly rolling back the finger

from the hole of the stopper, then replacing the finger when the

5

starch solution recedes. An overheated starch solution bubbles up

easily. Swirling the starch flask helps to settle the solution and aids

in release of air bubbles. Degasing is complete when no small

bubbles remain in the starch solution or on the side of the flask;

only the large boiling bubbles will remain. The finger is lifted

gently off the hole of the stopper to prevent re-entry of air bubbles

into the gel.

The starch solution is then poured into the gel mold (Fig. 2).

Pouring is done fairly rapidly to reduce the possibility of creating

air bubbles. Any air bubbles, lumps, or contaminants can be removed with a pipet before the starch solution begins to cool and set

up. The solution can be poured onto the gel strips to increase the

thickness of the gel and maximize the number of usable slabs after

electrophoresis. In approximately 45 min, when the gel feels cool

to the touch, it is ready to be used. Placing the gel briefly in a
refrigerator can speed cooling. Gels can usually be made I day in

advance with no noticeable effect. 2 To prevent desiccation, gels

are covered with plastic wrap after cooling and stored at room

temperature (storage in a refrigerator can alter physical properties

of the gel, making them unsuitable for subsequent electrophoresis).

Application of enzyme extracts to a gel _

Before the enzyme extracts of the tissue subsamples are applied to

a gel, the gel must undergo some initial preparation. A scalpel is

drawn vertically around the inside edge of the gel mold and the

long strips are removed. Using a sharp, clean scalpel, the gel is cut

from side to side, with the aid of a ruler, about 3 cm from the edge

of one of the long sides of the gel. The exact location of this

incision can vary under special circumstances, depending upon the

relative mobility of the enzyme being studied. Care must be taken

to ensure a smooth, vertical cut. This cut is referred to as the

origin-the location on the gel where enzyme extracts are applied.

The narrow strip of gel is called the cathodal strip, and the remaining portion is the anodal piece. The cathodal strip is gently pulled

away from the anodal piece using the fingertips until an approximately I-cm gap is obtained at the origin. The gel, still on the glass

plate, is placed on a repackaged, frozen Blue Ice package [(DIVASEX, Tustin, CA) Table II and is now ready for extract application

(Fig. 4).

The frozen tissue subsamples in the test tubes are centrifuged at

2000 x g at room temperature (22°C) for 15 to 20 min. We use a

Sorvall RT6000 centrifuge with the H-lOOOB swinging bucket

rotor (DuPont Co., Wilmington, DE). This process thaws the

lThe loss of visible electrophoretic varialion was observed for pelagic arrnorhead

(Pen/aceros richardsoni) when the gel had been prepared more than 12 h beforehand. This unusual phenomenon occurred only for the dimeric EST-D locus using

liver tissue and the TBCL gel buffer.

tissue and provides a fairly clear enzyme extract. The tubes are

transferred from their storage rack into the centrifuge racks for the

centrifuge process. After centrifugation, the centrifuge racks with

the tubes are placed in an icewater bath or on a frozen Blue Ice

package to keep the samples cold during the application process.

The enzyme extract from each test tube is applied to the gel

using paper wicks. We make wicks 3-4X 13 mm from heavy-duty

filter paper (Schleicher and SchueII No. 470). A wick is picked up

with forceps and dipped into a tube (Fig. 5). The wick draws up

the extract and is removed from the tube when the wick is soaked

to about three-fourths of its length. The reason for not soaking the

wick its full length is that a completely saturated wick will lose

some extraction fluid when touched to the gel and glass plate. This

excess fluid will "run together" with adjacent wicks and adversely

affect their electrophoretic patterns. However, it is desirable to

introduce as much of the enzyme extraction fluid to the gel as

possible. Therefore, close attention is needed to ensure proper

soaking of the wicks.

One way to ensure proper soaking of wicks is to observe the

reflection of light on the surface of each wick. Just after a wick is

removed from a tube, extraction fluid will be present on the surface

of the wick, as indicated by the reflection of light on the surface

of the wick. As the fluid spreads to the remaining quarter of the

wick, the surface fluid is drawn into the wick and light reflection

disappears. If this reflection does not disappear, then the wick is

oversoaked and some fluid is removed by touching the wick to a

paper towel. If the reflection disappears before the full length of

the wick is soaked, then additional fluid is drawn up the wick to

maximize the amount of sample applied to the gel.

Wicks are placed vertically in the origin on the anodal portion

of the gel from left to right (Fig. 4). The wicks will adhere to the

gel surface which will aid in their application. The first wick is

placed about 5 mm from the left inside edge of the gel. Subsequent

wicks are placed in the origin with a separation of about I mm. It

is important to make sure that each wick is vertical, that it touches

Figure 4

Preparing the gel for application of enzyme extracts and applying wicks to a gel. The gel (la and lb) resting

on a frozen Blue lee package (2) is separated at the origin (3). Wicks (4) are placed about 1 mm apart; wicks

with marker dyes are placed every 10 specimens. The wide portion of the gel is the anodal piece (1a); the

narrow portion is the cathodal strip (lb).

6
Figure 5

Drawing up the extract onto the wick. Samples are held in an icewater bath (I) or on a Blue Ice package

during the wick application process. For each sample, a wick (2) is dipped into a tube (3), allowing tbe extract

to be soaked up, which then is applied to a gel (Fig. 4).

the glass plate, and that adjacent wicks do not touch each other.

After each set of 10 wicks, a wick dipped in a red tracking dye is

applied. 3 Dye markers provide a standardized perspective of how

far enzymes have migrated in a gel and aid in recording data.

Initially, it may be helpful to apply the dye markers first before

applying enzyme extracts to aid in proper spacing of wicks. We

routinely apply 40 to 50 extracts plus dye markers to a gel. Since

the dye can interfere with migration of some enzymes, we use

narrower wicks for dye markers (I - 2 x 13 mm) and increase the

space on either side of each marker.

When all the wicks have been applied to the origin, the cathodal

gel strip is pushed up close to, but not touching, the wicks and

carefully aligned with the anodal piece. This alignment is important because the cathodal strip will stretch when it is moved. If the

cathodal strip were in contact with the wicks with this tension, it

could cause the wicks to twist out of position as the strip shifted to

relieve the tension. After alignment, the cathol strip to pushed up

to make firm contract with the wicks and anodal piece. The inward

pressure should be enough to cause the wicks to move with the

pressure. The wicks viewed through the gel should have a uniform

color. A circular spot of a different shade is evidence of an air

pocket. Air pockets disrupt electric current and should be removed

by applying localized pressure on the cathodal strip. To prevent

desiccation, a piece of plastic wrap, slightly larger than the gel, is

placed on the gel surface. A portion of the plastic wrap is folded

back exposing approximately 10 mm of the gel surface along the

length of both sides of the gel.

3We use a dye solution containing both cathodal and anodal migrating components.

The dye consists of one part red food coloring, containing FD and C Red Nos. 3 and

40 (Cresent Mfg. Co., SeaRle. WA), and two parts of a histological dye called

Fuschin Red (Pararosaniline. Sigma Chemical Co., SI. Louis, MO) made up in a

1.0% solution and adjusted to pH 7.0 with 1.0 M NaOH. The two red components

in the food coloring migrate anodally. One of these components migrates faster than

all enzymes normally detected, and therefore is used to detect the end point of

electrophoresis. The other red component migrdtes approximately one-half as far

anodally and is useful in recording data. The Fuschin Red migrates cathodally and

therefore is useful as a visual marker for cathodally migrating enzymes.

7

Setting up a gel for electrophoresis _

When the gel is ready for electrophoresis, the gel and glass plate

are taken off the Blue Ice package and placed on a cO'oling plate.

A cooling plate is part of a self-contained chilled water-circulation

system (Fig. 6). The purpose of the system is to remove heat

generated during electrophoresis and keep the gel cold, minimizing

enzyme degradation. Alternatively, the gel may be cooled on a

Blue Ice package or in a refrigerator. An electrode buffer tray is

placed on either side of the cooling plate. We use "organizer trays"

(Rubber Maid Inc., Wooster, OH), 23x7 cm, cut down to a height

of 3 cm. Approximately 250 mL of electrode buffer (Appendix 2)

is used for each tray. The connection between the electrode buffer

and the gel is made using absorbent sponge cloths [(e.g., Everywhere Wiper, Lola Products Corp., Hackensack, NJ) Table 1].

The cloths are allowed to absorb the electrode buffer in the buffer

tray, then they are placed on the surface of the exposed portions of

both sides of the gel and tucked in along both venical edges of the

gel. Moderate pressure is applied to the cloths to ensure good

contact (Fig. 7). The gel is connected to a DC power supply

(capable of an output of 400 V) using two electrodes. 4 The electrode connected to the positive terminal is placed in the electrode

buffer adjacent to the anodal piece. The electrode connected to

the negative terminal is placed in the electrode buffer adjacent to

the cathodal strip. Electrodes are held in place with wooden clips

(Fig. 7).5

4The electrodes are made from a patch cord having a banana plug at each end. The

cord is cut in half and a portion of the exposed wire is wrapped around a 3-cm piece

of 20-gauge platinum wire. Platinum wire is necessary because of its resistance to

corrosion during electrophoresis. A short piece of flexible tubing, which fits snugly

around the patch cord, is pushed over the wire covering the connection. Epoxy is

used to fill the open end of the tubing which seals the connection from possible

corrosion during electrophoresis but leaves at least 2 cm of exposed platinum wire.

5With this electrophoresis setup there is the possibility of electrical shock. Therefore,

it is necessary to exercise caution when working with or near gels during electrophoresis.
Figure 6

Cooling system for gels with five cooling plates (J). A cooling plate is a watertight box with internal channels causing water to circulate into and out of the plate (see

Table I for dimensions). The top surface of the plate is aluminum to facilitate heat transfer. A thin sheet of plastic covers the aluminum surface to insulate it from the

electrical current. Each plate is connected to a 2"C water circulating chiller (2) via a system of PVC pipe and insulated tubing (3) which pumps water through the system.

A valve (4) at the end of the PVC pipe system controls the amount of water flow through the plates. Arrows indicate direction of flow.

Current is now applied to the gel. The voltage and milliamp

settings will vary depending upon the buffer. For the three buffers

we commonly use (see Appendix 2), AC and TBCL gels run at

250 V, whereas a TBE gel runs at 350 V. Current is never allowed

to go higher than 70 mA to prevent the buildup of excess heat,

which would result in suboptimal electrophoretic separation. The

current is applied for 15 min with the wicks in place. After this

time, the power is turned off and the wicks are removed (it is

necessary to remove the wicks because in time the wicks begin to

dry out, which will hinder current flow across the gel).

Removing the wicks and reassembling the gel to continue electrophoresis are critical steps. Proper attention and care are necessary to ensure straight, consistent migration of enzymes in the gel

matrix. The power is turned off and the electrode buffer cloth on

the cathodal side is folded back into the buffer tray. The plastic

wrap is folded back to expose the origin and wicks. The cathodal

strip is pulled ftway from the wicks. Using forceps, the wicks are

carefully removed and discarded. Bits of the wick paper will often

stick to the gel. The origin must be very clean for current to flow

evenly across the gel. To ensure a clean origin, tips of the forceps

are dipped into the electrode buffer and then carefully run along

both sides of the origin, picking up bits of filter paper along the

way. It may be necessary to repeat this step several times to get the

origin clean. The cathodal strip is then pushed back up firmly

against the anodal strip, taking care not to trap air between the two

sides of the gel at the origin. To minimize the occurrence of air

pockets, start at one end of the cathodal strip, or in the middle, and

push it up to the anodal piece at a wide angle to the origin (as

opposed to no angle when you push the cathodal strip up to the

anodal piece when the wicks are still in place). As before, trapped

air pockets can be seen as a change in color at the origin. These

pockets can easily be removed by gently pulling the surface of the

gel away from the origin. After contact is made, the cathodal strip

can be moved from side to side to line it up properly with the

anodal piece (as opposed to prealigning the cathodal strip when the

wicks are still in place). The plastic wrap and cloth are then

replaced and the current reapplied to the gel.

The amount of time for an electrophoretic run depends upon the

buffer type and the amount of separation that is needed for the

enzymes of interest. Typically, we run AC and TBE gels until the

fast red dye migrates to the far edge of the anodal piece (approximately 10 cm from the origin). This takes about 4 h for an AC gel

8

and about 5 h for a TBE gel. A TBCL gel has a visible "boundary"

which migrates through the gel; the fast red dye migrates with this

boundary zone. We usually run the fast red dye in a TBCL gel until

it has moved 6 to 7 cm, about 3.5 to 4 h.

Slicing a gel _

Following completion of electrophoresis, the gel is sliced horizontally into a number of thin slabs. Each slab can be individually

stained for a different enzyme. Prior to slicing, the gel and glass

plate are removed from the cooling system and placed on a firm

foundation. The plastic wrap and short plastic strips are removed.

The comers of the gel on the end where the first wick was applied

are cut off at an angle. This is done to keep track of the direction

of extract application on the gel. A narrow sliver is cut off the

entire side of the other end of the gel so that the leading edge for

slicing will have a consistent, moist texture. Also, a cm or more

of gel may be cut off the entire length of the outside of the anodal

edge to facilitate accurate slicing when it is known that no enzymes

of interest have migrated that far.

Before slicing the gel, it is necessary to determine if the gel is

adhering to the glass plate. Gels can collect moisture between the

glass plate and the gel, causing them to slide off the plate during

slicing. To obviate this problem, both the cathodal strip and anodal

piece are carefully removed, placed briefly on paper towels, and

then repositioned on a dry glass plate. After repositioning, any air

bubbles beneath the gel are gently pressed out.

The gel and glass plate are then returned to their foundation for

slicing and positioned as illustrated in Figure 8. Two plastic

"slicing" strips (see Table I) are placed on the glass plate along

both long sides of the gel. To keep the slicing strips from sliding

off during slicing, they may be placed so that they rest against the

technician, or they may be clamped to the glass plate. The gel is

sliced by pulling a piece of nylon thread or 2-lb test monofilament

fishing line (approximately 90 cm long) through the gel, using the

slicing strips for adjusting the height of the slice through the gel.

The ends of the thread are wrapped securely around both index

fingers with enough thread between fingers to span the width of the

gel. With both index fingers pointed toward the technician, the

thumbs are placed on the thread and pressed onto the two slicing

strips above the top edge of the gel as shown in Figure 8. The
Figure 7 A gel setup for electrophoresis. Electrode bulTer trays (I) are on each

side of the gel (2) which is on a cooling plate (3). Electrodes (4) are held

in the electrode bulTer with wooden clips. Absorbent cloths (5) connect

the electrode bulTer to the gel. The cathodal (-) and anodal (+) portions

of the gel are determined by their connection to the power supply (6).

thread is entered at a slight angle to the leading edge of the gel and

drawn through at a slow but constant rate. Constant tautness on the

thread and slight downward pressure on the slicing strips are maintained. The amount of tautness and pressure needed to ensure

accurate slicing can be learned only by experience. Only a slight

arc in the thread should be present as it passes through the gel. If

too little tension is applied, the thread will fluctuate down to a

lower slice in the gel, affecting the smooth slicing of both slabs.

If too much tension is applied, the thread will break; if this happens, the slice is simply done again with another piece of thread.

After making the slice, another slicing strip is placed on top of each

of the previous slicing strips and the process repeated until the top

of the gel is reached. The first and final slices are often the most

difficult. so the thread is drawn through extra slowly to achieve

smooth slices. Up to seven or eight usable slabs can be cut from

a single 300-mL gel in this manner. Plastic slicing strips up to 2

mm thick are sometimes used when fewer slabs are needed, or

when a thicker slab gi ves greater levels of enzyme activity.

Staining a gel _

The slabs of a sliced gel are now prepared for staining. The top slab

has a rubbery upper surface due to its exposure to the air and

always gives very poor enzyme resolution. Therefore, this slab is

peeled off and set aside (it is possible to slice and remove the top

9

slab prior to electrophoresis which can help migration and resolution of the enzymes as well as decrease the time of electrophoresis). Subsequent slabs are peeled off and individually placed on

glass plates (Table 1). The cathodal strip may be placed and

aligned with the anodal piece depending upon location of the

enzyme of interest. The thin slabs are sometimes difficult to handle

at first because they are easily torn and slightly tacky. One method

for handling them is to get the fingertips of one hand under one end

of the gel near the edge, gently lift the slab all the way off, and then

get the fingertips of the other hand under the end of the slab that

is hanging. Others use a method by which both hands hold one end

of the gel while the other end is left to hang down. A spatula can

help in separating the slabs. Some find it useful to wet their fingers

and the glass plate to aid in the handling of the slabs. Slabs torn

during handling can usually be pieced back together and stained.

The upper slabs of the gel provide poorer enzyme resolution than

lower slabs of the gel due to the top rubbery slab. Thus, the first

two usable slabs next to the rubbery top slab are inverted, when

laid out on the glass plate. to expose the lower surface of the gel

for staining. When laying out the slabs, it is best to avoid trapping

air bubbles under the gel slab as they can affect the even distribution of the stain. Each slab is appropriately labeled with population

name. sample numbers, buffer type, and enzyme abbreviation

using transparent tape applied to the glass plate.

To stain each gel slab, a mixture of a hot agar solution and a

staining reagent is poured over the gel slab. We use a 2% agar

solution for staining, i.e., 2 g agar dissolved by heating in 100 mL

of distilled water. The desired amount of agar is sifted into heated,

distilled water which is just about to boil. The solution is swirled

frequently as the agar is added, to minimize the possibility of

creating lumps. The solution is kept on "low" heat with intermittent swirling until no granules are visible in the solution. Once it

is melted, the agar solution is held as a liquid at 65°C in a hot water

bath. Hardened agar is reused by remelting the agar in a boiling

water bath. A microwave oven works well in dissolving agar as

well as in remelting hardened agar. We use granular agar (BactoAgar, Difco Laboratories, Detroit, MI), as opposed to a powdered

agar, because it melts faster and produces a clear solution.

A staining reagent, in general, consists of a buffer of an appropriate pH containing the substrate for a particular enzyme, any

cofactors or enhancers that are required for that particular enzyme,

and a dye. 6 Typically, the dye precipitates or changes color in the

presence of one of the products of the enzyme reaction. In some

cases, the site of enzyme activity can be detected by the lack or

presence of fluorescence under an ultraviolet light. In either case,

the results of staining are visible bands where the enzyme is present

in the gel slab. Interpretation of these banding patterns is thoroughly covered in Utter et al. (1987).

To cover one gel slab, 25 mL of the staining mixture (three parts

staining reagent and two parts liquid agar) are needed. When

pouring this mixture over the gel surface, a spatula is sometimes

used to help spread the staining mixture evenly over the gel and to

hold back or remove bubbles. After a minute or less. the agar will

set up or gel. and the stained gel slab on the glass plate can be put

into an incubator. Incubation at 37°C is used for most enzymes to

speed up enzyme reaction and limit exposure to light (some of the

dyes that are used are reactive to light). Incubation time varies

depending upon the enzyme: some enzymes produce bands almost

6A number of chemicals used in the stains. particularly the dyes, are hazardous and

should be handled with cau'ion. It is advisable to (I) measure out the hazardous

chenlicals in a hood to minimize the breathing of dust and (2) wear disposable gloves

when preparing or pouring stains and in handling the stained gel plates. Hazardous

chemicals are usually readily identified with warning labels on the container.
Figure 8

Slicing a gel. The gel (I) has been moved onto a dry glass plate (2) and positioned so the edge with the cut

corners (3) is toward the technician. A slicing strip (4) is placed along both sides of the length of the gel. A

piece of nylon thread, wrapped around both index fingers and held taut, is pulled along the surface of the

two slicing strips to slice the gel. Two more slicing strips are added, and the slicing process repeated until

the top of the gel is reached.

immediately, whereas others take 30 min or longer before any

activity is seen. Thus, it is important to monitor the progress of

each stain so that no gels are allowed to "overstain" before the

results can be recorded. The results of the enzyme stains can be

preserved by photographing those gels which reveal data of particular interest. Stain preparation procedures and complete staining

recipes for 47 enzymes are given in Appendix 1.

Acknowledgments _

We thank the many reviewers for their time and constructive

thoughts on this manuscript, Laurie Barber and Linda Carlquist for

typing the manuscript, and Janice So for design and artwork of the

cover and Figure I.

Citations _

AEBERSOLD, P. B., G. B. MILNER, G. A. WINANS.

1986. Starch gel electrophoresis: the effect of tissue disruption procedures and

tissue dilution buffers on the stainin~ activity of specific enzymes. Unpubl.

manuser., Northwest Alaska Fish. Cent., Natl. Mar. Fish. Serv., NOAA,

2725 Montlake Blvd. E., Seanle, WA 98112.

AVISE, J. C.

1974. Systematic value of electrophoretic data. Syst. Zool. 23:465-481.

AYALA, F. J.

1976. Molecular evolution. Sinauer Assoc. Inc., Sunderland, MA, 277 p.

BOYER, S. H., D. C. FAINER, and M. A. NAUGHTON.

1963. Myoglobin: inherited structural variation in man. Science 140:1228-

1231.

BREWER, G. J., AND C. F. SING.

1970. An introduction to isozyme techniques. Academic Press, Inc., New

York, 186 p.

CLAYTON, J. W., and D. N. TRETIAK.

1972. Amine-citrate buffers for pH control in starch gel electrophoresis. J.

Fish. Res. Board Can. 29:1169-1172.

10

HARRJS, H., and D. A. HOPKINSON.

1976. Handbook of enzyme electrophoresis in human genetics. American

Elsevier, New York.

HARTL, D.

1980. Principles of population genetics. Sinauer Assoc. Inc., Sunderland,

MA, 488 p.

INTERNATIONAL UNION OF BIOCHEMISTRY.

1984. Enzyme nomenclature 1984. Academic Press, Inc., Orlando, FL,

646 p.

MAY, B., J. E. WRIGHT, and M. STONEKJNG.

1979. Joint segregation of biochemical loci in salmonids: results from experiments with Sa/veUnum and review of the literature in other species. J. Fish.

Res. Board Can. 36: 1114-1128.

NEVO, E., A. BElLES, and R. BEN-SHLOMO.

1984. The evolutionary significance of genetic diversity: ecological, demographic and life history correlates. In Mani, G. S. (editor), Evolutionary

dynamics of genetic diversity, p. 13-213. Lecture Notes Biomath. 53,

Springer-Verlag, New York.

RIDGWAY, G. J., S. W. SHERBURNE, and R. D. LEWIS.

1970. Polymorphism in the esterases of Atlantic herring. Trans. Am. Fish.

Soc. 99:147-151.

SHAKLEE, J. B., and C. P. KEENAN.

1986. A practiCal laboratory guide to the techniques and methodology of electrophoresis and its application to fish fillet identification. CSIRO Marine

Laboratories Publ. 177, Melbourne, Australia, 59 p.

SICILIANO, M. R., and C. R. SHAW.

1976. Separation and visualization of enzymes on gels. In Smith, I. (editor),

Chromatographic and electrophoretic techniques, Vol. 2, p. 185-209. Zone

electrophoresis. Heinemann, London.

SMITH, I.

1968. Techniques of starch gel electrophoresis. In Smith, I. (editor), Chromatographic and electrophoretic techniques, Vol. 2, p. 217-238. Zone

electrophoresis. Interscience Publishers, John Wiley and Sons, Inc., New

York.

UTTER, F. M., H. O. HODGINS, and F. W. ALLENDORF.

1974. Biochemical genetic studies of fishes: potentialities and limitations. In

Malins, D. (editor), Biochemical and biophysical perspective in marine biology, Vol. I, p. 213-237. Academic Press, San Francisco.

UTTER, F. M., P. B. AEBERSOLD, and G. A. WINANS.

1987. Interpreting genetic variation detected by electrophoresis. In Ryman,

N., and F. M. Utter (editors), Population genetics and fishery management,

p. 21-45. Sea Grant Publications, Seattle, WA.
APPENDIX 1: Staining recipes _

Following is a list of staining recipes for enzymes which we use in

our electrophoretic studies of fishes. They are adapted from recipes

given in Harris and Hopkinson (1976) and Siciliano and Shaw

(1976) using the agar overlay method. They are divided into four

categories to facilitate stain preparation: stains involving NAD+

dependent enzymes, stains involving NADP+ dependent enzymes,

enzymes detected with ultraviolet light, and miscellaneous stains.

For each enzyme stain, the full enzyme name is given as well as

an abbreviation, the presumed or observed subunit structure of the

enzyme when known, and the number assigned to the enzyme by

the Enzyme Commission (E.C.) of the International Union of

Biochemistry (1984). When preparing the stains, items can be

premixed on the day to be used except for those preceded by an

asterisk, which should be added just prior to staining. Items are

listed in order of most common use. Stain buffers, which are

usually the first item, are made at a given concentration and adjusted to the desired pH with the item following the slash (I). For

HCI, we use a 12.1-M concentrated solution, and for citric acid a

1.0-M solution. All phosphate buffers are made by mixing appropriate quantities of monobasic, dibasic, or tribasic sodium phosphate 0.1 M solutions to achieve the desired pH. Items which are

listed as percent solutions (g per 100 mL) are dissolved in distilled

water unless indicated otherwise. To expedite the preparation of

stains, it is useful to rely on an "estimated volumetic unit" (EVU)

as opposed to weighing out each chemical, and to use drops of

coupling enzymes from a pipet by calculating the number of units

in a drop. EVU equivalents are given next to milligram quantities

and relate to the approximate volume these quantities yield when

measured by eye on the tip of a spatula.

ADP

ATP

DMSO

E.C.

EVU

FAD

GAPDH

G6PDH

GDH

HCI

LDH

MgCl2

MIT

NAD+

NADH

NADP+

NADPH

NaOH

PMS

XO

List of abbreviations

Adenosine diphosphate

Adenosine triphosphate

Dimethyl sulfoxide

Enzyme Commission, IntI. Union Biochem.

Estimated volumetric unit

Flavine adenine dinucleotide

Glycerilldehyde-3-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase

Glutamate dehydrogenase

Hydrochloric acid

Lactate dehydrogenase

Magnesium chloride

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

f3-Nicotinamide adenine dinucleotide

f3-Nicotinamide adenine dinucleotide (reduced)

f3-Nicotinamide adenine dinucleotide phosphate

f3-Nicotinamide adenine dinucleotide phosphate

(reduced)

Sodium hydroxide

Phenazine methosulfate

Xanthine oxidase

I1
Stains Involving NAD+ Dependent Enzymes

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Glyceraldehyde-3-phosphate dehydrogenase GAPDH

tetramer E.C. 1.2.1.12

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Alanine aminotransferase

dimer

0.1 M Phosphate, 0.1 % 0.-

Ketoglutaric acid, pH 8.5

NAD+ 0.5%

ADP

Pyridoxal-5-phosphate

L-Alanine

*GDH (suspended in 50% glycerol

with negligible ammonium ions)

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Alcohol dehydrogenase

dimer

0.2 M TfI5/HCl, pH 8.0

NAD+ 0.5%

Ethyl alcohol 100%

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Fructose-biphosphate aldolase

tetramer

0.2 M Tris/HCl, pH 8.0

NAD+ 0.5%

Arsenic ac!d (sodium salt)

Fructose-I, 6-diphosphate

*GAPDH

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Fumarate hydratase

tetramer

0.2 M Tris/HCl, pH 8.0

NAD+ 0.5%

Pyruvic acid (sodium salt)

Fumaric acid (sodium salt)

*Malate dehydrogenase

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

ALAT

E.C.2.6.12

15 mL

0.5 mL

7 mg (1/2 EVU)

0.5 mg (1/50 EVU)

20 mg (112 EVU)

100 units

0.5 mL

0.5 mL

10 mL

ADH

E.C. 1.I.l.I

15 mL

0.5 mL

0.08 mL

0.5 mL

0.5 mL

IOmL

FBALD

E.C.4.1.I.l3

15 mL

0.5 mL

50 mg (1 EVU)

28 mg (1 EVU)

200 units

0.5 mL

0.5 mL

10 mL

FH

E.C. 4.2.1.2

15 mL

0.5 mL

5 mg (1/4 EVU)

50 mg (I EVU)

300 units

0.5 mL

0.5 rnL

IOmL

Glutamate dehydrogenase

hexamer

0.1 M Phosphate, pH 8.5

NAD+ 0.5%

ADP

L-Glutamic acid (sodium salt)

*Pyridoxal-5-phosphate

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

0.2 M Tris/HCI, pH 8.0

NAD+ 0.5%

Arsenic acid (sodium salt)

Aldolase

Fructose-I, 6-diphosphate

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Glycerol-3-phosphate dehydrogenase

dimer

0.2 M Tris/HCl, pH 8.5

NAD+ 0.5%

a-Glycerophosphate

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Hydroxyacylglutathione hydrolase

dimer

0.2 M TrisIHCI, pH 8.0

Methyl glyoxal

Glutathione (reduced)

Lactoyl-glutathione lyase (GLOI)

*NAD+ 0.5%

*Pyruvic acid (sodium salt)

*1.0 M Zinc chloride

*LDH

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

GDH

E.C. 1.4.1.2

15 mL

0.5 mL

7 mg (1/2 EVU)

45 mg (I EVU)

0.5 mg (1150 EVU)

0.5 mL

0.5 mL

10 mL

15 mL

0.5 mL

50 mg (1 EVU)

20 unils

28 mg (1 EVU)

0.5 mL

0.5 mL

10 mL

G3PDH

E.C. 1.1.1.8

15 mL

0.5 mL

35 mg (I EVU)

0.5 mL

0.5 mL

10 mL

HAGH

E.C.3.1.2.6

15 mL

0.24 mL

25 mg (I EVU)

150 units

3 mL

5 mg (114 EVU)

0.08 mL

400 units

0.5 mL

0.5 mL

10 mL

Incubate at 37°C in the dark until sufficient activity is present.

12

Rinse all glassware used in stain with distilled water prior to

use.

Mix first four items at least 30 min prior to staining.

Incubate at 37°C in the dark until sufficient activity is present.
L-iditol dehydrogenase

tetramer

IDDH

E.C. 1.1.1.14

Triosephosphate isomerase

dimer

TPI

E.C. 5.3.1.1

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

TAT

E.C. 2.6.1.5

15 mL

0.5 mL

25 mg (1/2 EVU)

15 mL

0.5 mL

7 mg (112 EVU)

8 mg (114 EVU)

0.2 mL

0.5 mg (1150 EVU)

100 units

0.5 mL

0.5 mL

10 mL

10 mg (112 EVU)

200 units

0.5 mL

0.5 mL

IOmL

0.03 M Tris, 0.005 M Citric acid,

0.6 mM Lithium hydroxide,

3.0 mM Boric Acid (TBCL gel buffer

diluted 1 in 10, see Appendix I),

pH 8.7

NAO+ 0.5%

Arsenic acid (sodium salt)

Oihydroxyacetone phosphate

(lithium salt)

*GAPOH

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

0.2 M Tris/HCI, pH 8.5

NAD+ 0.5%

ADP

ex-Ketpglutaric acid

L-Tyrosine/HCI 5.0% in 1.0 M HCI

*Pyridoxal-5-phosphate

*GOH (suspended in 50% glycerol

with negligible ammonium ions)

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Tyrosine aminotransferase

subunit structure uncertain

MDH

E.C. 1.1.1.37

LDH

E.C. 1.1.1.27

2 mL

0.5 mL

0.5 mL

10 mL

13 mL

0.5 mL

2 mL

0.5 mL

0.5 mL

10 mL

13 mL

0.5 mL

13 mL

0.5 mL

2 mL

0.5 mL

0.5 mL

IOmL

Malate dehydrogenase

dimer

0.2 M Tris/HCI, pH 8.0

NAO+ 0.5%

0.5 M Malic acid/NaOH (-1.0 M),

pH 7.0

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Lactate dehydrogenase

tetramer

0.2 M Tris/HCI. pH 8.0

NAO+ 0.5%

0.5 M Lactic acid/NaOH (-0.5 M),

pH 8.0

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

0.2 M Tris/HCI, pH 8.5

NAO+ 0.5%

Sorbital50.0%

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Incubate at 37°C in the dark until sufficient activity is present.

Stains Involving NADP+ Dependent Enzymes

Aconitate hydratase

monomer

AH

E.C. 4.2.1.2

Adenylate kinase

monomer

AK

E.C. 2.7.4.3

Incubate at 37°C in the dark until sufficient activity is present.

1.0 M Tris/HCl, pH 8.0

NAOP+ 0.25%

1.0 M MgCI 2

Aconitic acid

*Isocitrate dehydrogenase

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

15 mL

0.5 mL

I mL

12 mg (1/2 EVU)

5 units

0.5 mL

0.5 mL

10 mL

0.2 M Tris/HCl, pH 8.0

NAOP+ 0.25%

1.0 M MgCI 2

G6PDH

Glucose

ADP

*Hexokinase

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

15 mL

0.5 mL

0.25 mL

12 units

100 mg (2 EVU)

14 mg (I EVU)

1.5 mg (1/4 EVU)

0.5 mL

0.5 mL

IOmL

Incubate a 37°C in the dark until sufficient activity is present.

13
Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

lIn fish, the skeletal muscle CK isozymes act as monomers.

Incubate at 3rC in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Creatine kinase

dimer1

0.2 M Tris/HCI, pH 8.0

NADP+ 0.25%

1.0 M MgCl2

G6PDH

Glucose

ADP

Phosphocreatine

*Hexokinase

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Glucose-6-phosphate dehydrogenase

dimer

0.2 M Tris/HCI, pH 8.0

NADP+ 0.25%

1.0 M MgCI2

Glucose-6-phosphate

*MTT 0.5%

"PMS 0.5%

*Agar 2.0%

Glucose-6-phosphate isomerase

dimer

0.2 M Tris/HCl, pH 8.0

NADP+ 0.25%

1.0 M MgCI 2

G6PDH

Fructose-6-phosphate

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Hexokinase

monomer

0.2 M Tris/HCl, pH 8.0

NADpr 0.25%

1.0 M MgCI2

G6PDH

Glucose

ATP

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

CK

E.C. 2.7.3.2

IS mL

0.5 mL

0.25 mL

12 units

100 mg (2 EVU)

14 mg (I EVU)

18 mg (I EVU)

1.5 mg (1/4 EVU)

0.5 mL

0.5 mL

IOmL

G6PDH

E.C. 1.1.1.49

15 mL

0.5 mL

0.25 mL

50 mg (2 EVU)

0.5 mL

0.5 mL

IOmL

GPI

E.C. 5.3.1.9

IS mL

0.5 mL

0.25 mL

12 units

5 mg (1/4 EVU)

0.5 mL

0.5 mL

10 mL

HK

E.C.2.7.1.1

15 mL

0.5 mL

0.25 mL

12 units

100 mg (2 EVU)

14 mg (J EVU)

0.5 mL

0.5 mL

10 mL

Isocitrate dehydrogenase

dimer

0.2 M Tris/HCI, pH 8.0

NADP+ 0.25%

1.0 M MgCl2

Isocitric acid (sodium salt)

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Malate dehydrogenase (NADP+)

tetramer

0.2 M Tris/HCI, pH 8.0

NADP+ 0.25%

1.0 M MgCI2

Oxaloacetic acid

0.5 M Malic acidiNaOH (-1.0 M),

pH 7.0

*MTT 0.5%

*PMS 0.5%

"Agar 2.0%

Mannose-6-phosphate isomerase

monomer

0.03 M Tris, 0.005 M Citric acid,

0.6 mM Lithium hydroxide,

3.0 mM Boric acid (TBCL gel buffer

diluted I in 10, see Appendix I.),

pH 8.7

NADP+ 0.25%

1.0 M MgCI2

G6PDH

Mannose-6-phosphate

*Glucose-6-phosphate isomerase

*MTT 0.5 %

*PMS 0.5%

*Agar 2.0%

Phosphoglucomutase

monomer

0.2 M Tris/HCl, pH 8.0

NADP+ 0.25%

1.0 M MgCI2

G6PDH

Glucose-I-phosphate (with

I% glucose-I, 6-diphosphate)

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

IDH

E.C. 1.1.1.42

IS mL

0.5 mL

0.25 mL

36 mg (I EVU)

0.5 mL

0.5 mL

10 mL

MDHp

E.C. 1.1.1.40

13 mL

0.5 mL

05 mL

9 mg (1/4 EVU)

2 mL

0.5 mL

0.5 mL

IOmL

MPI

E.C. 5.3.1.8

IS mL

0.5 mL

0.25 mL

12 units

7 mg (I EVU)

10 units

0.5 mL

0.5 mL

10 mL

PGM

E.C. 5.4.2.2

15 mL

0.5 mL

0.1 mL

6 units

13 mg (1/2 EVU)

0.5 mL

0.5 mL

10 mL

14

Incubate at 37°C in the dark until sufficient activity is present.
Phosphogluconate dehydrogenase

dimer

0.2 M Tris/HCI, pH 8.0

NADP+ 0.25%

1.0 M MgCI2

6-Phosphogluconic acid

(ammonium salt)

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

PGDH

E.C. 1.1.1.44

15 mL

0.5 mL

0.25 mL

20 mg (1 EVU)

0.5 mL

0.5 mL

10 mL

Incubate at 37°C in the dark until sufficient activity is present.

Enzymes Detected with Ultraviolet Light

Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

~-N-Acetylgalactosaminidase

dimer

0.1 M Phosphate/Citric acid, pH 9.5

*4-Methylumbelliferyl-N-acetyl-[3-Dgalactosaminide (dissolve first in

0.25 mL DMSO)

*Agar 2.0%

N-Acetyl-~-glucosaminidase

dimer

0.1 M Phosphate/Citric acid, pH 4.5

*4-Methylumbelliferyl-N-acetyl-[3-Dglucosaminide (dissolve first in

0.25 mL DMSO)

*Agar 2.0%

Acid phosphates

dimer

~GALA

E.C.3.2.1.53

15 mL

5 mg (1/4 EVU)

10 mL

~GA

E.C. 3.2.1.30

15 mL

5 mg (114 EVU)

10 mL

ACP

E.C. 3.1.3.2

Enolase

dimer

0.2 M Tris/HCl, pH 8.0

ADP

Fructose-I, 6-diphosphate

1.0 M MgCI2

Potassium chloride

2-Phosphoglyceric acid (sodium salt)

*NADH

*Pyruvate kinase

*LDH

*Agar 2.0%

Esterase-D

dimerJ

0.1 M Phosphate, pH 7.0

4-Methylumbelliferyl acetate

(or butyrate) (dissolve first in

0.5 mL acetone)

*Agar 2.0%

ENO

E.C. 4.2.1.11

15 mL

14 mg (1 EVU)

7 mg (1/4 EVU)

0.5 mL

165 mg (3 EVU)

12 mg (1 EVU)

30 mg (3 EVU)

200 units

400 units

10 mL

EST-D

E.C. 3.1.1.*

15 mL

1.5 mg (1/4 EVU)

10 mL

Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

0.1 M Phosphate/Citric acid,

pH 3.5 or 6.5

*4-Methylumbelliferyl phosphate

*Agar 2.0%

15 mL

4.5 mg (1/2 EVU)

IOmL

15

Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

lIn addition to a unique dimeric locus, this stain also detects a

class of enzymes which does not represent a single ancestral

lineage; therefore subunit structure may vary among loci.
Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

a-Glucosidase

tetramer

0.1 M phosphate/Citric acid. pH 8.0

*4-Methylumbelliferyl-a-D-glucoside

*Agar 2.0%

a-Mannosidase

subunit structure uncertain

0.1 M Phosphate/Citric acid, pH 9.5

*4-Methylumbelliferyl-a-D-mannopyranoside (dissolve first in 0.25 mL

DMSO)

*Agar 2.0%

aGLU

E.C. 3.2.1.20

15 mL

3 mg (1/4 EVU)

10 mL

aMAN

E.c. 3.2.1.24

15 mL

6.5 mg (1/4 EVU)

IOmL

Phosphoglycerate kinase

monomer

0.2 M Tris/HCl, pH 8.0

1.0 M MgCI2

ATP

3-Phosphoglyceric acid (sodium salt)

*NADH

*GAPDH

*Triosephosphate isomerase

*Glycerol-3-phosphate dehydrogenase

*Agar 2.0%

Pyruvate kinase

tetramer

PGK

E.C. 2.7.2.3

15 ml

I mL

14 mg (I EVU)

55 mg (I EVU)

30 mg (3 EVU)

375 units

300 units

75 units

10 mL

PK

E.C. 2.7.1.40

Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

u.2 M Tris/HCI, pH 8.0

ADP

Fmdose-I, 6-diphosphate

1.0 M MgCI 2

Potassium chloride

Phospho(enol)pyruvate

*NADH

*LDH

*Agar 2.0%

15 mL

14 mg (1 EVU)

7 mg (1/4 EVlJ)

0.5 mL

165 mg (3 EVU)

12 mg (1 EVU)

30 mg (3 EVU)

400 units

IOmL

Incubate at 37°C, until sufficient activity is visible under longwave (365 nm) UV light.

Miscellaneous Stains

Incubate at 37°C in the dark until sufficient activity is present.

Adenosine deaminase

monomer

0.1 M Phosphate, pH 8.5

Arsenic acid (sodium salt)

Adenosine

*XO

*Purine-nucleoside phosphorylase

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

ADA

E.C. 3.5.4.4

15 mL

50 mg (I EVU)

3 mg (1/4 EVU)

3 units

5 units

0.5 mL

0.5 mL

IOmL

Aspartate aminotransferase

dimer

0.12 M Tris, 0.02 M Citric acid,

2.4 mM Lithium hydroxide,

12.0 mM Boric acid (TBCL

gel buffer diluted 1 in 5,

see Appendix 1),

0.22% a-Ketoglutaric acid.

0.5% L-Aspartic acid, pH 7.5

(adjust pH with 3.0 M Potassium

hydroxide)

"'Fast blue BB salt

"Agar 2.0%

AAT

E.C. 2.6.1.1

15 mL

17 mg (1/2 EVU)

10 mL

Incubate at 37°C in the dark until sufficient activity is present.

16
Carboxylesterase

subunit structure variableI

0.1 M Phosphate, pH 7.0

*(X-Napthyl propionate (dissolve

first in 4.0 mL acetone)

*Fast blue BB salt

EST

E.C. 3.1.1.1

75 mL

12.5 mg (1/2 EVU)

34 mg (I EVU)

General protein stain

Glacial acetic acid

Ethyl alcohol 100%

Distilled water

Naphthol Blue Black 1%

Nigrosin 1%

I part

5 parts

4 parts

GP

Place gel slab in a staining tray, pour staining solution into tray,

then agitate tray to loosen gel slab from the bottom of tray.

Place gel slab in a staining tray, pour enough staining solution

into the tray to cover the gel (approximately 75 mL), and then

agitate the tray to loosen the gel slab from bottom of the tray.

Change the destain periodically, as it turns blue, until all excess

stain has been removed.

Incubate at 22°C for approximately 15 min, then pour the staining solution off (save for repeated use), rinse gel slab with

water, and then cover with destain solution (first three items).

Incubate at 22°C until sufficient activity is present, pour off the

staining solution, and rinse the gel slab in water.

Other possible substrates are (X-Naphthyl acetate and

(X-Naphthyl butyrate.

IThis stain detects a class of enzymes which does not represent

a single ancestral lineage; therefore subunit structure may vary

among loci.

13-Glucuronidase

tetramer

13GUS

E.C. 3.2.1.31

Appears immediately as yellow zones on blue-green background.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Incubate at 37°C in the dark until sufficient activity is present.

Catalase

tetramer

Hydrogen peroxide 0.06%

Apply to filter paper overlay as

needed to keep moist and incubate at 22°C for 5 min; remove

filter paper, soak up excess

H20 2 solution, and add the following:

*Potassium ferricyanide 2.0%

(make the same day as used)

*Ferric chloride 2.0% (make the

same day as used)

*Agar

Diaphorase

subunit structure uncertain

0.2 M Tris/HCl, pH 8.0

2, 6-Dichlorophenol-indophenol 0.02%

in 0.2 M Tris/HCl, pH 8.0

*FAD

*NADH or NADPH

*MTT 0.5%

*Agar 2.0%

CAT

E.C. 1.11.1.6

10 mL

7 mL

7 mL

IOmL

DIA

E.C.*.*.*.*.I

13 mL

2 mL

2 mg (1/8 EVU)

7.5 mg (1/2 EVU)

2 mL

10 mL

0.2 M sodium acetate/HCI, pH 5.0

*Naphthol-AS-BI-(3-D-glucuronide

*Fast garnet GBC salt

*Agar 2.0%

Glutathione reductase

dimer

0.2 M Tris/HCI, pH 8.0

2, 6-Dichlorophenol-indophenol 0.02%

in 0.2 M Tris/HCI, pH 8.0

Glutathione (oxidized)

*FAD

*NADH

*MTT 0.5%

*Agar 2.0%

Guanine deaminase

dimer

0.2 M Tris/HCI, pH 8.0

Guanine 2.0% in 1.0 M NaOH

*XO

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

15 mL

10 mg (1/2 EVU)

10 mg (1/2 EVU)

10 mL

GR

E.C. 1.6.4.2

13 mL

2mL

18 mg (I EVU)

2 mg (1/8 EVU)

7.5 mg (1/2 EVU)

2mL

10 mL

GDA

E.C. 3.5.4.3

15 mL

0.1 mL

3 units

0.5 mL

0.5 mL

IOmL

IThis stain may detect Xanthine oxidase or a variety of other

enzymes.

17

Incubate at 37°C in the dark until sufficient activity is present.
Lactoyl-g1utathione lyase

dimer

LGL

E.C. 4.4.1.5

Purine-nucleoside phosphorylase

trimer

PNP

E.C. 2.4.2.1

Incubate at 37°C in the dark until sufficient activity is present.

O. I M Phosphate, pH 7.0

Methyl glyoxal

Glutathione (reduced)

Mix above items, apply to filter

paper overlay as needed to keep

moist, and incubate at 37°C for

at least 45 min; remove filter

paper and add the following:

*Iodine 2.0%/Potassium iodide 4.0%

(make the same day as used)

*Agar 2.0%

IS mL

1.0 mL

48 mg (2 EVU)

1.0 mL

24 mL

0.1 M Phosphate, pH 8.5

Arsenic acid (sodium salt)

Inosine

*XO

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

Superoxide dismutase

dimerl

15 mL

50 mg (I EVU)

72 mg (3 EVU)

3 units

0.5 mL

0.5 mL

10 mL

SOD

E.C. 1.15.1.1

12mL

500 mg

1201L

15 mL

I mL

0.12 mL

24 mg (I EVU)

IThe mitochondrial form of this enzyme is a tetramer.

Incubate at 37°C in the dark until activity just begins to appear

and then set in mdirect light until sufficient activity is present.

XO

E.C. 1.2.3.2

15 mL

0.5 mL

0.5 mL

10 mL

IS mL

0.2 mL

0.5 mL

0.5 mL

10 mL

Xanthine oxidase

subunit structure uncertain

0.2 M Tris/Hel, pH 8.5

*MTf 0.5%

*PMS 0.5%

*Agar 2.0%

0.2 M Tris/ HCI, pH 8.0

Hypoxanthine 4.0% in 1.0 M NaOH

*MTT 0.5%

*PMS 0.5%

*Agar 2.0%

~TP

E.C. 3.6.1.19

Appears immediately, then fades.

Nucleosidetriphosphate pyrophosphatase

dimer

0.2 M Tris/HCI, pH 8.0

1.0 M MgCI2

2-Mercaproethanol

Inosine triphosphate

Mix above items, apply to filter

paper overlay as needed to keep

moist, and incubate at 37°C for

at least I h; remove filter paper

and add the following:

*Molybdic acid (ammonium salt)

2.5% in 2.0 M sulfuric acid

*Ascorbic acid tsodium salt)

*Agar 2.0%

Incubate at 37°C in the dark until sufficient activity is present.

Peptidase

subunit structure variable'

PEP

E.C.3.4.*.*

Incubate at 37°C in the dark until sufficient activity is present.

0.1 M Phosphate, pH 7.5 (use pH

6.5 for L-Phenylalanylglycylglycyl-L-phenylalanine peptide)

O-Dianisidine (dihydrochloride)

Snake venom (Western Diamondback

Rattlesnake)

Peroxidase

*Peptide substrate. e.g., Glycyl-Lleucine, DL-Leucylglycylglycine,

L-Leucyl-L-tyrosine,

L-Phenylalanyl-L-proline,

L-Phenylalanylglycylglycyl-Lphenylalanine

*Agar 2.0%

IS mL

6 mg (1/4 EVU)

4 mg (1/4 EVU)

2 mg (1/2 EVU)

20 mg (I EVU)

IOmL

Incubate at 37°C in the dark until sufficient activity is present.

IThis stain detects a class of enzymes which does not represent

a single ancestral lineage; therefore subunit structure may 'Vary

among loci.

18
APPENDIX 2: Electrophoresis butTers _

Following are recipes for our three most commonly used gel

buffers. The first two are continuous buffer systems (where components of the gel buffer are the same as components of the electrode buffer), and the third is a discontinuous buffer system (where

components of the gel buffer are different than components of the

AC : Amine, Citric acid butTer (Clayton and Tretiak 1972)

Electrode, pH 7.0*:

Citric acid 0.04 M

N-(3-aminopropyl)-morpholine: add until pH is reached

EDTAI (optional) 0.01 M

Gel: use 1 in 20 dilution of the electrode buffer.

*For certain circumstances where it is desirable to have the

locus of interest migrate more toward the cathode, a pH of 6.0

or 6.5 is sometimes used.

lEthylenediarninetetraacetic acid (sodium salt).

TBE : Tris, Boric acid, EDTA butTer (Boyer et al. 1963)

Electrode, pH 8.7:

Tris 0.18 M

Boric acid 0.1 M

EDTA 0.004 M

Gel: use 1 in 4 dilution of the electrode buffer.

19

electrode buffer). Recipes for the buffers are presented in units of

molarity (M) or moles per liter. To calculate the number of grams

of a chemical required in a specific volume of buffer, multiply the

molarity (miL) times the formula or molecular weight (g/m) of the

chemical times the desired volume (L).

TBCL: Tris, Citric acid gel butTerlLithium hydroxide,

Boric acid electrode butTer (Ridgway et al. 1970)

Electrode, pH 8.0:

Lithium hydroxide 0.06 M

Boric acid 0.3 M

EDTA (optional) 0.01 M

Gel, pH 8.7:

Tris 0.3 M

Citric acid 0.05 M

Add 10% of the electrode buffer.

Use 1 in 10 dilution of the gel buffer.