Isoelectric Focusing ofProteins on Agarose Gels

Section XII: Isoelectric Focusing of Proteins on Agarose Gels

In This Section
Introduction
Preparation of Agarose Isoelectric Focusing Gels
Running Agarose IEF Gels
Staining Proteins in Agarose IEF Gels
Press Blot Transfer of Proteinsfrom Agarose IEF Gels
Preparative Isoelectric Focusing
Immunofixation/Immunoperoxidase
or Autoradiography
Direct Tissue Isoelectric Focusing
Resolution Reference Guide
References

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190
191
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Section XII: Isoelectric Focusing of Proteins on Agarose Gels

Introduction

Compatible agaroses

Separation of proteins in complex mixtures for analyticalresolution can be achieved by isoelectric focusing (IEF),
in which proteins are separated based on their net charge(isoelectric point, or pI) in the presence of a pH gradient.
Analytical focusing is carried out either in polyacrylamidegels—most recently prepared with immobilized pH
gradients—or in agarose gels prepared with mobile carrierampholytes, which form a pH gradient when subjected toelectrophoresis. Separation in agarose gels is more rapid,
since the pores of the agarose gel are larger than those ofpolyacrylamide gels.

Isoelectric focusing on agarose gels requires the use ofan agarose that has no measurable electroendosmosis(EEO). Lonza has developed two products that can beused for this application, specifically IsoGel® Agarose and
IsoGel® Agarose IEF Plates.

■■ Advantages—— No toxic monomer solutions are required—— IsoGel® Agarose is a highly purified agarose that iseasy to prepare and produces a less restrictive gelthan polyacrylamide, allowing rapid focusing of highmolecular weight proteins (>2,000 kDa).
—— IsoGel® Agarose IEF Plates are ready-to-use 125 mm
x 100 mm precast gels that eliminate gel preparation
time and provide easy handling throughout IEF
processing.

—— Separation of proteins larger than 2,000 kDa

Isoelectric Focusing ofProteins on Agarose Gels—— Shorter staining times—— Nontacky and compressible (blottable)—— No catalysts to interfere with separation■■ Applications

The photograph below demonstrates the separation
performance of proteins focused on an IsoGel® Agarose
IEF Plate.
Alternatively, Lonza offers IsoGel® Agarose IEF Precast
Plates.

—— Immunofixation directly in the gel—— Crossed immunoelectrofocusing—— Direct tissue isoelectric focusing—— Preparative isoelectric focusing

1

2

3

4

5

Proteins
Lentil lectin

Equine myoglobin –Bovine carbonic
anhydrase –Ov albumin –Amyloglucosidase –Separation of proteins in a IsoGel® Agarose IEF Plate, pH 3-10. Lanes 1& 4: in-house pI Marker. Lanes 2 & 3: Broad Range pI 4.45-9.6 marker(BioRad). Lane 5: Hemoglobin, HB Type AFSC (PE Wallac). 2.5 µl of eachsample were loaded on the gel and prefocused at 1 watt for 10 minutes
and focused at 2000 volts (max), 25 mA (max), 25 W (max) for 60
minutes on a GE Healthcare MultiPhor® II chamber at 10°C. The gel was
stained with Crowle’s Stain.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Preparation of Agarose Isoelectric Focusing Gels

Suggested anolytes and catholytes

Preparation of the gel casting assembly

When selecting anolytes and catholytes for any pH
gradient, it is important to closely bracket the endsof the pH range of the ampholytes. Avoid creating pH
discontinuities between the ends of the ampholyte range
and the bracketing electrolytes.

1.

Spread a few drops of distilled water or 0.1% nonionicdetergent on one glass plate.

2.

L ay a sheet of GelBond® Film, cut slightly smaller than
the glass plate, onto the plate with the hydrophilic sideup.

 ote: pH is dependent on temperature. 25ºC pH valuesNare provided for selection of electrolytes. Under running
conditions, the pH will be slightly higher.

Note: Water droplets spread on the hydrophilic side but bead
up on the hydrophobic side of the film.

		AnolyteConcentration 		 pH (25ºC)
1M
1.0
Phosphoric acidSulfuric acidAcetic acidL-glutamic acid
Indole acetic acid

0.2 M
0.5 M
40 mM
3 mM

1.6
2.6
3.2
3.8

L-tyrosine

4 mM

4.5

		
Catholyte
Concentration 		 pH (25ºC)
Threonine
50 mM
5.8
Glycine
50 mM
6.15
Hepes
0.4 M
7.3
L-Histidine (free-base)*
40 mM
7.35
Bicine0.1 M
8.0
Sodium hydroxide
1M
13.0*Do not substitute Histidine HCl for free-base.

3.

Cover the GelBond® Film with a sheet of blotting paper orthe interleaving paper supplied with the film.

4.

Firmly roll with a rubber roller or wipe with tissues tosqueeze out any air bubbles and excess fluid from behind
the GelBond® Film.

5.

Carefully wipe off any excess liquid at the edges.

6.

Place the U-frame spacer on top of the GelBond® Film. If a
U-frame spacer is unavailable, place two spacers on the
GelBond® Film along either side and one spacer acrossthe bottom edge.

7.

Place the second glass plate over the spacer(s).

8.

Clamp the assembly with the stationery clamps, using 2
clamps per side and bottom.

9.

Warm the cassette in a 55ºC forced hot air oven for 15
minutes.

Isoelectric Focusing ofProteins on Agarose Gels

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Note: GelBond® Film may warp if the cassette is heated too
long or above 75ºC.

■■ Materials–– GelBond® Film (110 mm x 125 mm, Cat. No. 53745)–– Blotting paper or interleaving paper supplied with
Gelbond® Film–– Two thick glass plates (110 mm x 125 mm)–– A plastic 0.8 mm-thick U-frame spacer or 3 single
spacers, 0.8 mm-thick–– Six stationery binder clamps–– Blotting paper, rubber roller or tissues–– Forced hot air oven set to 55ºC■■ Reagents–– Distilled water or 0.1% nonionic detergent

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Preparation of Agarose Isoelectric Focusing Gels — continued

The following procedure is to prepare a 10 ml IsoGel®
Agarose gel.
Solution preparation■■ Materials–– Erlenmeyer flask or beaker (50 ml)–– Microwave–– Boiling water bath or hot plate1.

Choose a 50 ml beaker or Erlenmeyer flask.

2.

Add 8 ml distilled water and a stir bar to the flask or
beaker.

–– Magnetic stir bar

3.

Premeasure 0.1 g IsoGel® Agarose.

–– 1-cc tuberculin syringe

4.

Premeasure 1.0 g d-sorbitol.

–– Water bath set to 60°C

5.

Sprinkle in the premeasured agarose powder, while thesolution is rapidly stirring.

–– 20-cc syringe

Using a spatula, break up and disperse any agarose
clumps and scrape down any powder adhered to thewalls of the flask.

–– Parafilm® or tape

6.

Isoelectric Focusing ofProteins on Agarose Gels

7.

Add the d-sorbitol while the solution is rapidly stirring.

8.

Remove the stir bar.

9.

Follow the procedures on page 83 for dissolving
agarose.

10.

Cool the solution to approximately 60ºC.

11.

Add 0.63 ml of ampholyte solution with a 1-cc syringe.

12.

Stir the solution well to mix.

13.

Maintain the agarose solution at 60°C - 65ºC until
casting.

14.

Correct for evaporation by adding warm distilled water–– Magnetic stir plate–– Prewarmed cassette assembly–– Spatula■■ Reagents–– IsoGel® Agarose (see page 56)–– Distilled water–– Ampholytes–– D-sorbitol–– Boiling distilled water–– 60°C distilled water

immediately before gel casting.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Preparation of Agarose Isoelectric Focusing Gels — continued

Gel casting instructions
1.

Flush a 20 cc syringe with boiling water to thoroughly
heat it.

2.

Expel all water from the barrel and needle.

3.

Immediately fill the syringe with the warmed agarose
solution.

4.

Slowly inject the agarose solution into the pre-warmedcassette.

Note: Try to avoid introducing air bubbles into the cassette by
injecting the solution in a slow-steady stream.
5.

Fill the cassette assembly to the top with agarose
solution.

6.

Seal the top of the cassette with Parafilm® or tape toprevent evaporation.

7.

Allow the casting assembly to cool at room
temperature.

8.

Place the gel at 4ºC for one hour.

Disassembly of casting cassette1.

Remove the tape and clamps.

2.

With the cassette lying flat, insert a flat spatula between
the glass plates.

3.

Twist gently to break the seal.

4.

Carefully remove the top plate, leaving the gel and the
GelBond® Film attached to the back plate.

5.

Remove the spacers.

6.

Lift the GelBond® Film from the back plate by inserting aflat spatula under the film.

7.

Gently lift film and gel away from glass plate.

Sample Preparation
Successful isoelectric focusing, in part, depends upon thecondition of the sample. Situations such as insolubility or
high salt content, particularly in the case of high sample
loading, should be addressed before the sample is loaded
onto the gel. Listed below are general guidelines forsample treatment.
High Salt Content: Dialyze the sample against distilledwater, 1% glycine, or 0.05 M - 0.1 M ammonium bicarbonate
solution.
Dissociation of protein aggregates, subunit assemblies or
to unfold peptide chains: Add urea to a final concentrationof 4 M - 9 M to both the sample and the gel.
Samples that are hydrophobic or poorly soluble at or near
their pI point:
Add either nonionic or zwitterionic detergents to thesample and the gel at a final concentration of 0.05% - 1.0%.
Detergents should be added to the agarose solution once
the agarose has been dissolved and cooled to 60°C.
Nonionic detergents
Triton® X-100

Nonidet® (NP-40)

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Zwitterionic detergentsCHAPS (available from
Sigma Chemical Co.,
St. Louis, MO)
Zwittergent® 3-14
(available from Calbiochem/		Behring, LaJolla, CA)

Tween® 80		

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Running Agarose IEF Gels

Caution: Make certain the power supply is turned off
before proceeding.

■■ Materials–– Horizontal IEF chamber–– Fine-grained blotting paper

Procedure for gel placement–– Razor blade1.

Set the refrigerated circulator bath to 10°C - 15ºC.

–– Refrigerated circulator bath set to 10°C - 15°C

Note: To prevent condensation on the gel and platen, do not 		
circulate the coolant to the IEF chamber just prior to focusing.

–– Kimwipe® Tissues or equivalent–– Electrode wicks

Isoelectric Focusing ofProteins on Agarose Gels

2.

Spread 0.2 ml - 0.3 ml of distilled water on the coolingplaten of the IEF chamber.

3.

L ower the gel (film-side down) onto the wetted area.
Avoid trapping air under the GelBond® Film.

–– Forceps

4.

Wipe excess fluid from the edges of the film.

–– Sample applicator mask

5.

Blot the surface of the gel briefly with a sheet of finegrained blotting paper.

–– Power supply

6.

If necessary, trim the edges of the gel parallel to thedirection of focusing with a razor blade to ensure theedges are even and free of cracks or small tears.

–– Scissors–– Glass plate slightly larger than the gel■■ Reagents–– Distilled water–– Catholyte solution–– Anolyte solution

Procedure for electrode wick application
1.

C ut two electrode wicks to the exact width of the gel orslightly shorter.

2.

Completely immerse one wick in catholyte solution.

3.

Place the wick on blotting paper to remove excess fluid.

4.

Place on the negative electrode contact of the gel.

5.

Completely immerse a second wick in anolyte solution.

6.

Place the wick on blotting paper to remove excess fluid.

7.

Place on the positive electrode contact of the gel.

Procedure for sample application
1.

Place the sample applicator mask across the gel at least
1 cm from either wick (e.g., 3 cm from cathode).

2.

L oad sample and pI markers into the slots (2 µl - 5 µl
maximum; 2 - 10 mg/ml concentration).

Note: In direct tissue isofocusing, tissue samples may be 		placed directly onto the applicator slots.
3.

E nsure electrodes and electrical contacts are clean andthere are no breaks in the wire or ribbon.

4.

P lace the electrodes on the wicks (not the gel surface),aligning them so they lie in parallel upon the wicks.

Place a glass plate on top of the gel and wicks forapproximately one minute.

5.

Set the power supply at 1 W constant power.

6.

Apply power for 10 minutes.

Note: This ensures uniformity of contact between wicks
and gel and serves to smooth the wick surface in 		
preparation for electrode contact.

7.

Turn power off.

8.

Remove the sample applicator mask.

9.

Gently remove any precipitated sample from the gel
surface with blotting paper.

Note: The wicks must lie parallel to each other on the endsof the gel, evenly touching the surface.
8.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Running Agarose IEF Gels — continued

IEF power settings and focusing time1.

Start circulation of the coolant to the IEF chamber.

2.

Set the voltage, current and power according to theappropriate running conditions listed in the table below.

3.

Once focusing is complete, turn off the power.

4.

Discard the wicks.

5.

Place the gel in fixative solution.

Note: The separation progress can be monitored by observing
the visible proteins in the pI markers coming into focus andnoting the decreasing rate of current flow on the power supply’s
milliampere indicator. Focusing is attained when the visible pI
markers are sharply resolved and the current has stopped
decreasing significantly (less than 1 mA in 10 minutes).

Running Conditions
Ampholyte
2.5 - 4.5

Voltage
pH Range
250 V

CurrentPower(limiting)		
MAX
10 W

3.5 - 9.5
500 V
MAX
10 W
3.5 - 9.51000 VMAX
25 W
3.5 - 9.5
1500 VMAX
25 W
4 - 6.5
500 V
MAX
10 W
				
				
5-8
500 V
MAX
10 W
				
				
*1M H PO can be replaced by 0.5 M acetic acid3

AnolyteCatholyte(upper limiting)		
*1 M H3PO4
0.05 M Threonine

Focusing Time(minutes)
90

0.5 M HOAc
1 M NaOH
0.5 M HOAc
1 M NaOH
0.5 M HOAc
1 M NaOH
2% solution
0.04 M Lof pH 2.5 - 4.5
Histidine
Ampholyte(free-base)
2% solution
0.1 M NaOH orof pH 2.5 - 4.5
0.1 M Bicine
Ampholyte		

90
40
30

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90

90

4

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Staining Proteins in Agarose IEF Gels

Introduction■■ Materials

Either Coomassie® Blue or Crowle’s Double Stain canbe used to stain IEF gels. Coomassie® stain is used
when increased sensitivity is desired, and Crowle’s
stain produces gels with clear background and sharp
resolution.
Staining proteins with Coomassie® Blue Stain or Crowle’s
Double Stain
Preparation of staining solutions
Fixative solution
180 ml30.0 g18.0 gMethanolTtrichloroacetic acid
Sulfosalicylic acid

Isoelectric Focusing ofProteins on Agarose Gels

Adjust volume to 500 ml with distilled water
Coomassie® Stain
1.0 g
Coomassie®
Brilliant Blue R-250
250 mlEthanol
90 ml
Glacial acetic acid
Adjust volume to 1 liter with distilled water
Coomassie® Destaining Solution
250 mlEthanol
90 ml
Glacial acetic acid
Adjust volume to 1 liter with distilled water
Crowle’s Double Stain
2.5 g
Crocein scarlet (C.I. 26905)
150.0 mg
Coomassie®
Brilliant Blue R-250
50 ml
Glacial acetic acid30.0 gTrichloroacetic acid
Adjust volume to 1 liter with distilled water
Crowle’s Destaining Solution
3 ml
Glacial acetic acid
Adjust volume to 1 liter with distilled water–– Flask–– Stir bar–– Magnetic stir plate–– Forceps–– Paper towel–– Whatman® 3MM chromatography paper–– 1 kg-2 kg weight–– Glass plate–– Forced hot air oven set to 50°C-55ºC–– Staining vessel–– Clamps■■ Reagents–– Methanol–– Trichloroacetic acid–– Sulfosalicylic acid–– Distilled water–– Coomassie® Brilliant Blue R-250–– Ethanol–– Glacial acetic acid–– Crocein scarlet (C.I. 26905)

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Staining Proteins in Agarose IEF Gels­— Continued

Follow the steps below to stain the gel after electrophoresis
using either Coomassie® Blue or Crowle’s Double Dtain.

Note: Drying usually takes less than 30 minutes.
15.

Stain with Coomassie® or Crowle’s double stain solution
for 15 - 30 minutes without agitation.

1.

Place the gel in Fixative solution for 10 minutes.

2.

Remove the gel and rinse the surface with distilledwater.

3.

Drain excess water.

16.

Remove the gel and rinse with distilled water.

4.

Place on a paper towel, gel side up.

17.

Place the gel in Destaining solution for 3 minutes.

5.

Pre-wet a piece of Whatman® 3MM chromatography
paper with distilled water.

18.

Briefly rinse again in distilled water.

6.

Place on gel surface.

19.

Clamp (gel side out), onto a glass plate to prevent curling
during drying.

7.

Overlay the blotting paper with four to six layers ofabsorbent paper toweling.

8.

Place a glass plate on top of the paper toweling.

9.

Place a 1 kg - 2 kg weight on top of the toweling for 10
minutes.

10.

Remove the weight, the glass plate, and the paper
toweling.

11.

Rewet the blotting paper thoroughly with distilledwater.

12.

Gently lift the blotting paper off gel surface.

13.

Wash the gel in distilled water for 5 minutes to remove
residual fixative and ampholytes.

14.

Dry the gel completely in a forced hot air oven (50°C 55ºC).

Note: Float gel-face down into the stain, so precipitatedstain will not settle on the gel surface.

Dry the gel in a forced hot air oven (50°C - 55ºC) for
approximately 15 minutes or dry at room temperature
overnight.
Note: Gel may crack if over dried by heating.
20.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Staining Proteins in Agarose IEF Gels — continued

Staining Proteins in Agarose IEF Gels with Silver Stain
A modified silver stain procedure has been developedfor use with agarose gels cast on GelBond® Film. Afterelectrophoresis, the gels are fixed, press blotted, andcompletely dried before staining. Perform all fixing andstaining steps in acid-cleaned (50% HNO3) glassware.
All washes are done with constant agitation in a volume
of at least 250 ml (gel volume:reagent volume = 1:22).
Coomassie® Brilliant Blue stained gels may be silver
stained after drying. In this case, proceed directly to
step 14.
Preparation of staining solutions

Isoelectric Focusing ofProteins on Agarose Gels

Fixative solution
180 mlMethanol
30.0 gTrichloroacetic acid18.0 gSulfosalicylic acid
Adjust volume to 500 ml with distilled water
Silver Stain Solution A
50.0 gSodium carbonate, anhydrous
in 1 liter distilled water (Stable for2 - 3 weeks at room temperature)
Silver Stain Solution B
	In the order given dissolve thefollowing reagents in 1 liter of distilledwater, while mixing rapidly.
2.0 g
Ammonium nitrate
2.0 g Silver nitrate
10.0 g Dodeca-tungstosilicic acid6.7 ml 37% formaldehyde(Stable 1 week at room temperature
stored in the dark)
Stop solution
1%
Acetic acid■■ Materials–– Paper towel–– Whatman® 3MM chromatography paper–– 1 kg-2 kg weight–– Glass plate–– Forceps–– Clamps–– Forced hot air oven set to 50°C-55°C–– Staining containers–– Beakers–– Magnetic stir plate–– Magnetic stir bar–– Acid-cleaned dish–– Orbital or rocking platform shaker■■ Reagents–– Methanol–– Trichloroacetic acid–– Sulfosalicylic acid–– Distilled water–– Glutaraldehyde–– Dithiothreitol (DTT)–– Anhydrous sodium carbonate–– Ammonium nitrate–– Silver nitrate–– Dodeca-tungstosilicic acid–– (Gallard-Schlesinger Cat. No. 305453)–– 37% formaldehyde–– Acetic acid
Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.
Caution: Acetic acid causes burns and respiratory
irritation. Take precautions to prevent expursure.
Caution: Glutaralhyde is toxic and must be handled
in a fume hood.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Staining Proteins in Agarose IEF Gels —­ continued

Follow the steps below to stain the gel after electrophoresis
using silver stain

22.

Rinse the gel in distilled water.

23.

Wipe any silver deposits from the back of the film.

1.

Place the gel in Fixative solution for 10 minutes. If gel isprestained with Coomassie® Blue and dried, proceed tostep 14.

24.

Allow to air dry.

2.

Place the gel on a paper towel, gel side up.

3.

Pre-wet a sheet of Whatman® 3MM chromatography
paper with distilled water.

4.

Place on gel surface.

5.

Overlay the blotting paper with four to six layers ofabsorbent paper toweling.

6.

Place a glass plate on top of the paper toweling.

7.

Place a 1 kg - 2 kg weight on top of the toweling for
10 minutes.

8.

Remove the weight, the glass plate, and the paper
toweling.

9.

Rewet the blotting paper thoroughly with distilledwater.

10.

Gently lift the blotting paper off the gel surface.

11.

 ash the gel in distilled water for 5 minutes to removeWresidual fixative and ampholytes.

12.

Clamp (gel side out), onto a glass plate to prevent
curling during drying.

13.

Dry the gel in a forced hot air oven (50°C - 55ºC) for
approximately 15 minutes or until dry.

Isoelectric Focusing ofProteins on Agarose Gels

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Note: Gel may crack if over dried by heating.
14.

Soak the dried gel in 2% glutaraldehyde for 10 minutes.

15.

Wash in distilled water for 10 minutes using mild
agitation.

16.

Soak the gel for 10 minutes in 0.01% (DTT)
dithiothreitol.

17.

Wash in distilled water for 10 minutes using mild
agitation.

18.

Pour an equal volume of Silver stain solution B into
vigorously stirring Silver stain solution A (75 ml B and
75 ml A for each gel to be stained).

19.

Transfer the solution to an acid-cleaned glass dishcontaining one gel.

20.

Stain the gel for 10 minutes with gentle agitation.

Note: There will be some background.
21.

Transfer the gel to a Stop solution and gently agitate for5 minutes.
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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Press Blot Transfer of Proteins from Agarose IEF Gels

Press blot transfer is a quick method of removing
proteins from agarose gels. The procedure involves
overlaying the gel with a buffer-soaked nitrocellulose
membrane covered by a thick filter pad and several
layers of dry paper toweling. The assembly is thencovered by a glass plate. After just 11/2 minutes of press
blot, approximately 20% of the proteins are transferredfrom the gel to the membrane. Up to 85% transfer can beachieved with a 35 - 40 minute blotting time. Transferred
proteins can be detected on the membrane and on thegel by standard methods.
Procedure
1.

Prepare Tris-saline buffer, pH 7.5.

2.

C ut one piece of nitrocellulose membrane and thickfilter paper to the same dimension as the gel.

■■ Materials–– Nitrocellulose membrane–– Thick filter paper–– Scissors–– Smooth-tipped forceps–– Container–– Paper towels–– Glass plate■■ Reagents–– Tris-saline buffer, pH 7.5–– 	(50.0 g Tris-HCl, 0.94 g Tris-Base, 58.48 g NaCladjust to 2 liters with distilled water)

Isoelectric Focusing ofProteins on Agarose Gels

Note: Wear gloves to prevent contamination by extraneous
proteins.
3.

Evenly wet the nitrocellulose membrane in the Trissaline buffer by holding one end of the membrane withsmooth-tipped forceps and lowering the other end into
the buffer container, dropping the membrane flat on thebuffer surface.

Note: The membrane must be completely saturated withbuffer.
4.

Remove the gel from the focusing chamber.

5.

Place on a flat surface, gel side up.

6.

P lace the buffer-soaked nitrocellulose membrane onto
the gel surface.

Note: Avoid trapping air bubbles between the gel and themembrane.
7.

Place one piece of buffer soaked filter paper on top of
the membrane.

8.

Place three layers of dry paper toweling on top of thefilter paper.

9.

Cover with a glass plate slightly larger than the gel
surface. No other weight is required.

10.

Press blot for 11/2 minutes or longer, as desired.

11.

Remove glass plate and discard the paper toweling andfilter paper.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Preparative Isoelectric Focusing

Separation of relatively large amounts of biologically
active macromolecules is possible by isoelectric focusing
in agarose. Typical high-yield recoveries of applied proteins
are obtainable with retention of biological activity. Thispreparative isoelectric focusing procedure is based on thework of Cantarow, et al. As much as 120.0 mg of protein
can be focused in 9.5 ml (0.75 x 10 x 11.5 cm) of 1%
IsoGel® Agarose containing 2.5% ampholytes.
Procedure
1.

Follow steps 1 - 14 for Gel Preparation on pages 179-181.
Also see Sample Preparation on page 181.

2.

Add the protein sample to the agarose solution oncecooled to 60ºC.

3.

Stir the solution well to mix.

4.

Cast the gel and disassemble the casting cassette
following the steps previously described on page 179.

5.

Place the gel on the 10º C cooling platen for 10 minutes(if gel has not already been chilled after casting).

6.

Focus following the steps previously described on pages
182-183.

■■ Materials–– Spatula–– 5-cc plastic syringe with an 18-gauge needle–– Clean centrifuge tube–– Test tube rocker at 4°C–– Centrifuge–– Serum separator–– Parafilm®
■■ Reagent–– Phosphate-buffered saline (PBS)

Isoelectric Focusing ofProteins on Agarose Gels

Returnto MainProcedure for recovering proteins from preparative gel
1.

E xcise the gel slice containing the protein of interest byusing a spatula to strip the agarose from the GelBond®
Film in 5 mm-wide slices.

2.

Place the agarose strip in a 5-cc plastic syringe fittedwith an 18-gauge needle.

3.

Macerate the gel slice by expelling it into a clean tube.

4.

Add 4 ml of phosphate-buffered saline (PBS) to themacerated gel.

5.

Cover the tube securely with Parafilm®.

6.

Place on a test tube rocker for 16 hours at 4ºC.

7.

Centrifuge the tube for one minute at 100 rpm.

8.

Separate the supernatant from the gel using a serum
separator.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Immunofixation/Immunoperoxidase or Autoradiography

Detection of separate species can be accomplishedby protein stains or by overlaying gels with specificantibody solutions coupled to enzymes that will
eventually produce a visible end product. The antibodyperoxidase conjugate system or autoradiography with
125I- labeled antibody are frequently used for this purpose.
Immunoperoxidase labeling of focused proteins is
performed according to Saravis, et al. After focusing, fixing,
and drying the gel, treat the gel as described below.
Procedure for Immunoperoxidase
Staining/Avidin-Biotin Modification

Isoelectric Focusing ofProteins on Agarose Gels

1.

Soak the gel in 3% hydrogen peroxide for 10 minutes.

2.

Rinse with distilled water.

3.

Soak the gel in 2.28% periodic acid for 5 minutes.

4.

Rinse with distilled water.

5.

Soak the gel in 0.02% sodium borohydride for 2
minutes.

6.

Rinse with distilled water.

7.

Place the gel in 0.05 M Tris-saline, pH 7.6 for 10
minutes.

8.

I ncubate the gel with 1:5 normal serum for 10 minutes(same species as secondary antibody).

9.

Incubate the gel for 2 - 4 hours at room temperature with
Anti-serum(primary antibody).

10.

Rinse the gel with 0.15 M PBS, pH 7.4 for 1 hour atroom temperature.

11.

Incubate the gel with Secondary* antibody (e.g., goatanti-mouse IgG) for 2 - 4 hours at room temperature.

Note: If the avidin/biotin modification is used, proceed withsteps 12 and 13 using biotinylated reagents (marked with * insteps 11 and 15).

12.

Rinse the gel with 0.15 M PBS for 1 hour at room
temperature.

13.

Incubate the gel with avidin solution (40 mg/gel) in PBS
for 1 hour at room temperature (stock solution of avidin
is usually 20 mg/ml).

14.

Rinse the gel with 0.15 M PBS for 1 hour at room
temperature.

15.

Incubate the gel with horseradish peroxidase* (33 mg/gel) for 2 hours at room temperature.

16.

Rinse the gel with 0.15 M PBS for 1 hour at room
temperature.

17.

Incubate the gel with diaminobenzidine (0.15 mg/ml)
and hydrogen peroxide (0.03%) in 0.01 M Tris 0.15 M
NaCl for 1 - 17 hours at room temperature.

18.

Rinse the gel in PBS and dry.

■■ Materials–– Staining containers■■ Reagents–– Hydrogen peroxide–– Distilled water–– Periodic acid–– Sodium borohydride–– 0.05 M Tris-saline (pH 7.6), normal serum	(same species as secondary antibody)–– Anti-serum (primary antibody)–– 0.15 M PBS (pH 7.4)–– Secondary antibody–– 20 mg/ml avidin solution–– Horseradish-peroxidase–– 0.15 mg/ml diaminobenzidine–– 0.01 M Tris/0.15 M NaCl

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Direct Tissue Isoelectric Focusing (DTIF)

This method employs the application of tissue or cellpellets directly onto the surface of the IEF gel without
concentration of samples, dialysis to remove salts, or thesalt extraction of soluble proteins from tissue. DTIF allows
more soluble protein per milligram of tissue to enter thegel than is recoverable by extraction procedures andminimizes denaturation of biologically active proteinsthat can be damaged by extraction.
1.

Prepare solutions and cast gels as previously describedon pages 179-181.

2.

Prepare for focusing as previously described on page
182.

3.

Place the sample applicator mask across the gel at least
1 cm from either wick (e.g., 3 cm from cathode).

4.

Drape a tissue slice over the open slot of the applicator
mask.

5.

Ensure the electrodes and the electrical contacts areclean and there are no breaks in the wire or ribbon.

6.

Place the electrodes on the wicks (not the gel surface),aligning them so they are in parallel upon the wicks.

7.

Set power supply at 1 W (constant power).

8.

Prefocus for 10 - 15 minutes to allow sample uptake.

9.

Turn power supply off.

10.

Remove the tissue slice and the applicator mask.

11.

Focusing is continued using standard conditions.

12.

After focusing is complete, the gel is fixed, stained anddried following standard procedures.

Isoelectric Focusing ofProteins on Agarose Gels

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Resolution Reference Guide

Phenomenon
Streaks or gaps perpendicular
to bands

IEF Band Appearance

Possible Causes• Filter or centrifuge sample before applying.

• Old or denatured sample• Replace sample.

• Isoelectric precipitation of sample
applied too close to pl point• Do not prefocus gel before
applying sample.
• Apply sample in different location.
• Use solubilizing additive, I.e., non-ionic,
Zwitterionic• Try different application location.

• Poor soluble sample, which precipitates

Fuzzy bands• Run incomplete• Focusing time too long
• Insufficient fixation

Skewed bands• Electrode not clean• Uneven electrode contact

Isoelectric Focusing ofProteins on Agarose Gels

Missing or faint bands• Increase fixation time. Be sure to place gel infixative immediately after IEF run.
• Rinse electrode with distilled water, then dry.

• Sample placed too close to edge of gel• Electrode wicks too short• Wicks should extend to edge of gel.

• Old or incorrect electrolyte solution• Make fresh solution.

• Overloading of sample• Decrease protein load.

• Excessive salt in sample• Reduce salt concentration by gel filtration or
dialyze against 1% glycine.

• Protein not denatured by fixative• Establish appropriate
denaturing conditions.
• Use alternative application site.

• Excessive salt in sample• Overloading with sample,
distorting pH gradient• Improper anolyte usedArc-shaped bands• Use pl marker to monitor run or measure pH
gradient before removing gel from platen.
• Decrease focusing time or power/voltage.

• Wicks should be evenly wetted and placed on
gel parallel to each other.
• Place electrodes firmly on wicks but not so
fluid is squeezed from wicks.
• Apply > 5 mm from edge of gel.

• Sample unstable at pH of siteof application
Wavy bands

Remedy• Particulates in sample• Reduce salt concentration by gel filtration or
dialyze against 1% glycine.
• Applying smaller sample volume.
• Replace anolyte.

• Old or incorrect electrolyte solutions• Make fresh solutions.

• Dirty electrodes• Rinse electrode with distilled water.

• Thin spot in gel• Reduce evaporation by keeping gel sealed until
ready for use.
• Keep chamber lid closed except whenhandling gel.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Resolution Reference Guide — continued

Phenomenon
Burning or sparking

IEF Band Appearance

Possible Causes• Electrode wicks too dry• Thin spot in gel• Using incorrect electrolyte solutions• Always use recommended solutions.

• Focusing too long

Sparking along edge of gel onto
cooling plate• Excessive power input• Check power supply settings.

• Excess moisture on the gel, or under gel• Remove excess moisture by gently
blotting gel.
• Cut electrode stripes to size of gel.

• Electrode strips overhanging ends of gel

Formation of ‘ditch’ ultimately
causing gel to burn out• Liquid expelled at sides of electrode due to
EEO flow of water to cathode• Excess electrode pressure when placing electrodes
on gel• Occasional blotting may be necessary.

• Gradient drift• Check purity or reagents.

• High current causing excessive heating
early in run• Uneven gel thickness due to gel drying out• Use a low power for first 10-15 minutes ofrun during sample uptake.
• Open foil package containing gel justbefore use.
• Initially blot only until blotting paper picks up
moisture.

• Overblotting of gel, causing gel to dry out
Sample smearing or precipitation• Use firm but light pressure.

• Sample overload• Decrease sample concentration.

• Sample aggregation• Solubilize sample with neutral surfactant
or 1% glycine.
• Change application site. Solubiling additives ordetergents may be necessary.

• Sample applied too near pl or at a pH where it isinsoluble
Incomplete Sample Uptake

Remedy• Anodal wick should be wet not dripping wet;cathodal wick should be damp.
• Reduce focusing time to minimum required
to obtain linear pH gradient.
• Keep gel sealed until ready for use.

• Sample application mask not left on long enough• Allow at least 10 minutes.

• Insolubles or aggregates in sample• Filter or centrifuge sample.

• Get too wet when sample applied• Blot gel before sample application.

• Sample applied too near pl• Change application site.

• At Cathode• Unblotted wick
• Overfocusing• Blot cathode wick with filter paper until
damp before application.
• Monitor run with pl markers.

• Localized elsewhere• Salt in sample• Detach sample before electrofocusing.

• Sample overload• Decrease sample concentration.

• Uneven electrode contact to wicks• Check location of electrodes.

• Electrode wicks too wet• Remove excess moisture. Wicks must be
blotted before use.
• Avoid this area for sample application.

Sample retained in sample
application well

Isoelectric Focusing ofProteins on Agarose Gels

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Fluid expression• General (over entire gel)• Excessive amount along
electrode wicks. Persists forduration of run• Spotted over gel• Local hot spot from spillage of electrodesolution onto gel• Insufficient/excessive cooling• Check coolant flow and temperature.

• Excessive power applied to gel• Check power supply settings.

• Gel not adequately blotted• Use blotting paper.

• High ambient humidity• Use tray with desiccant in chamber.

• Cathodal drift (pooling of fluid near cathode—oversaturating wick)• Blot pooled liquid of necessary.

• Reversed polarity of electrode wicks (lower pH atcathrode; higher at anode)• Check pH of wicks and polarity of plugs into
power supply. Reverse polarity if incorrect.

• Localized hot spots caused by bubbles between
gel film and chamber platen• Check for air bubbles—remove if present.

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Section XII: Isoelectric Focusing of Proteins on Agarose Gels
Resolution Reference Guide — continued

Phenomenon

IEF Band Appearance

Condensation inside chamber

Possible Causes

Remedy• Inefficient cooling• Adjust coolant temp. to 10° C.

• Voltage/power setting too high• Adjust to 1W for 1st 10 min. 25W limiting
thereafter (pH 3-10 e.g.).
• Desalt sample.
• Place humectant cartridge or desiccant
tray in chamber.

• Salt in sample• Humid ambient conditions
Get thinning• at electrode• Improper electrolyte solution• Check electrolyte solution.

• near of point of application• Sample overload• Decrease sample concentration.

• generalized• Get left out of cassette too long under low
humidity conditions• Leave gel in cassette until ready to use orplace in humidity chamber.

• Incorrect calibration of pH electrode• Get overfocused—excessive gradient drift• Check pH electrode.
• Do not exceed time required to form alinear pH gradient.
• Check water for heavy metals or ioniccontaminants.
• Check solution.

Uneven pH gradient/pH gradientdifferent than stated for ampholyte used

• Impure distilled water in sample orelectrolyte solutions• Incorrect electrolyte solution
pH gradient is not linear• Insufficient run time

pH gradient not attaining upper limits• Overfocusing• pH measurements may be wrong• Cathodal drift (entire gradient shifts
towards cathode)• Electrodes applied at the wrong
ends of the gel

pH range shifted toward cathodeIsoelectric Focusing ofProteins on Agarose Gels

Current increases with time

Diagonal band migration

Lateral band migration• Uneven electrode contact• Apply electrodes evenly on wicks.

• Too much pressure on electrodes• Uneven electrode placement• Adjust for light but firm contact.
• Ensure electrodes are parallel andperpendicular to the sample migration.

• Air pocket(s) trapped beneath mask

• Sample mask left on too long/ not long enough• Press gently to expel before sample
applied.
• Reduce sample volume, increase sample
concentration.
• Remove mask after 10 minutes at 1 watt.

• Run incomplete• Sample unstable at pH of application• Allow longer focusing time.
• Use alternative application site.

• Removal of ligands during focusing• Check protein for this possible
characteristic.

• Formation of complex aggregate• Use urea to treat sample.

• Too much sample applied to applicator mask

Sample “focuses” at wrong position

A supposedly “pure” sample focuses asmultiple bands

High background stain• Sample exists in various states of oxidation• Pretreat sample accordingly.

• Sample disassociates into subunits• Natural phenomenon.

• Sample has become denatured• Check procedures used to prepare sample.

• Residual ampholytes in gel• Follow recommended press blot
procedure.
• Extend time of fixing, rinsing anddestaining.
• Reduce concentration.
• Make fresh stain and be sure all dye is insolution.
• Float gel side down in stain for 15-30
minutes.
• Follow recommended press blot/rinsing
procedure.

• Concentration of stain too high• Stain is old• Gel allowed to settle in bottom of staining dishDelamination (peeling) of gel from film
support on staining after drying• Use IsoGel® pl markers to check progress.
Measure pH gradient before removing gel.
• Decrease runtime.
• Use surface pH electrode.
• Reduce run time (as much as possible) toattain desired gradient profile.
• Use recommended anolyte/catholyte
solution.
• Apply (+) electrode to anode. (-)
electrode to cathode.
• Reverse polarity• Residual fixative in dried gel

References
Allen, R.C., et al., Gel Electrophoresis and Isoelectric Focusing of Proteins: Selected
Techniques, Walter de Gruyter & Co., 1984.
Westermeier, R., Electrophoresis in Practice, 2nd edition, Wiley Company, 1997.
Coligan, J.E., et al., Current Protocols in Protein Science, Wiley Company, 1999.
Harper, D.L., Electrophoresis ’81, 205 - 212, 1981.
Saravis , C.A. and Zamcheck, N., J. Immunol.Methods, 29: 91 - 96, 1979.
Cantarow, W.D., et al., Electrophoresis, 3: 85 - 89, 1982.
Ebers, G.C., et al., J. Immunol. Methods, 37: 315 - 323, 1980.
Saravis, C.A., et al., Electrophoresis, 1: 191 - 193, 1980.
Saravis , C.A., et al., J. Immunol. Methods, 29: 97 - 100, 1979.

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