Lonza Rockland, Inc.
www.lonza.com
biotechserv@lonza.com
Tech Service: 800-521-0390
Customer Service: 800-638-8174
Document # 18120-0807-06
Rockland, ME 04841 USA

Isoelectric Focusing with IsoGel® Agarose IEF Plates pH 3-10

Introduction

Circulation BathIsoGel® Agarose IEF Plates pH 3-10 are prepared by custom
blending IsoGel® Agarose with ampholytes. They form a lessrestrictive matrix than polyacrylamide and are ideal for rapid
focusing of high molecular weight proteins (>200 kDa). They
are particularly well suited for the separation and analysis ofantibodies. IsoGel® Agarose is cast on GelBond® Film toprovide dimensional stability and ease of handling throughout
processing. Plate dimensions are 100 mm x 125 mm.

1.
2.

Isoelectric Focusing Procedure
NOTE: These instructions are meant for gels run in the portrait
orientation (10 cm wide and 12.5 cm long). Running the gelsin the Landscape orientation (12.5 cm wide and 10 cm long)
may alter sample resolution.

Storage•Store plates at 4°C

NOTE: Remove gel from refrigerator and use immediately.
Gels left at room temperature for extended periods may adhere
to the upper cassette plate when the cassette is opened.

Materials Required•
Sample Applicator Mask•
Electrode Wicks•
Blotting Paper
The above three items are included in the IsoGel® IEF Plate
Accessory Pack (catalog # 56014 or 56010)•
Horizontal Isoelectric Focusing Chamber•
Constant Power Supply•
Refrigerated Circulator Bath•Forced Air Oven set at 50°C to 55°C•
pI Marker Proteins•
Micropipetter and Pipette tips

NOTE: A frosted or crystalline background on a gel when first
opened may indicate the product was frozen. If this occurs
call Technical Service for assistance.

Removing IsoGel® Plate from Cassette
1.
2.

3.

Electrolyte Solutions Required••Anolyte:
Catholyte:

Set the temperature on the circulator bath so that theplaten temperature reaches 10°C-15°C.
Begin circulation to the IEF chamber 15 to 20 minutes
prior to focusing.

0.5 M Acetic Acid, pH 2.6
1 M NaOH, pH 13

Cut the foil storage bag and remove the plasticcassette.
With the opening on the cassette facing downward,
remove the cassette cover by placing a spatula tip
between the two halves of the cassette and twist.
Remove the gel by gently pushing a finger through theopening in the cassette bottom.

Gel Placement1.

Preparing Protein Samples
For protein samples containing salts, dialyze the protein
sample against deionized water, 1% glycine OR 0.05 M to0.1 M ammonium bicarbonate.

2.

For samples that are hydrophobic or poorly soluble, add either
nonionic or zwitterionic detergent to the sample at a final
concentration of 0.05% to 1.0%
•Nonionic detergents:•Triton® X-100•Nonidet® (NP-40)•Tween ® 80•
Zwitterionic detergents:•CHAPS®
•Zwittergent® 3-143.

4.
5.

1

Wipe any condensation off the cooling platen. The
surface of the electrophoresis platform should be
between 10°C to 15°C.
Spread a small volume (100 μl) of deionized water onto
the cooling platen of the IEF chamber. This will holdthe gel to the platen by capillary action.
Lower the gel on the wetted area. Avoid trapping air
between the GelBond® Film and platen. If bubbles
form, use the edge of a scalpel blade or thin spatula togently lift the edge of the gel, then lay the gel backdown on the platen to remove the bubble.
Wipe or blot excess fluid from the edges of the film.
Briefly blot the surface of the gel with a sheet of blottingpaper.

Electrode Wick Application
1.
2.

3.

4.
5.
6.
7.

Completely immerse a wick in the catholyte solution(1 M NaOH). This is the cathodal wick.
Remove excess fluid from the wick by placing it briefly
on blotting paper, then gently blot the top surface of thewick with blotting paper.
Repeat steps 1 and 2 with another wick using theanolyte solution (0.5 M Acetic Acid). This is the anodalwick.
Place the cathodal wick at the (-) electrode contact ofthe gel.
Place the anodal wick at the (+) electrode contact of thegel.
Verify that the wicks are parallel and in even, smoothcontact with the gel surface.
Lay a glass plate on top of both wicks for 10 to 15
seconds to compress the wicks evenly on the gelsurface.

Preparation of Coomassie® Staining Solutions
Coomassie® Blue Stain•
1.0 g Coomassie® Brilliant Blue R-250•
250 ml ethanol•
90 ml glacial acetic acid•
Fill to 1 L with deionized waterCoomassie® Destaining•
250 ml ethanol•
90 ml glacial acetic acid•
Fill to 1 L with deionized waterPreparation of Crowle’s Double Stain Solutions
Crowle’s Double Stain•
2.5 g Crocein Scarlet (C.I. 26905)•150.0 mg Coomassie® Brilliant Blue R-250•
50.0 ml glacial acetic acid•
3.0 g trichloroacetic acid•
Fill to 1 L with deionized water

Sample Application
1.
2.
3.

4.
5.

6.

Place the sample applicator mask across the gel parallelwith the wicks and at least 1 cm from either wick.
Pipette 2 μl to 5 μl of sample solution into the applicatormask slots.
Place electrode assemblies onto the wicks, and align theelectrodes so they are parallel with the gel.
Tip: To minimize excess pressure on the IsoGel®
Agarose IEF Plate, cut four, 4 cm long wicks. Place thewicks on the four corners of the electrophoresis platform,
approximately 1.5 cm in from the short sides of theplatform, lying parallel to the short sides of the platform.
Position the wicks so that they cushion the electrodes,
relieving pressure on the gel.
Set the power supply at 1 W constant power and apply
power for 10 minutes to prefocus the samples.
Turn off the power and remove the sample applicatormask. The applicator mask can be reused. Rinse with
deionized water prior to storing.
Gently remove any precipitated sample from the gel
surface with blotting paper.

Crowle’s Destaining•
Tap water

Gel Fixation
1.

2.
3.

Place the gel in fixative for 20 to 30 minutes.
(Rubbermaid® Servin’ Savers® Rectangle #3865 or
Rubbermaid® Servin Savers® 2 or 5 cup squarecontainers work well.)Remove the gel from the fixative by grasping onto theedge of the gel film with forceps.
Place the gel on a paper towel, gel side up.

Gel Drying
1.

Wet a single sheet of Whatman® 3MM blotting paper with
deionized water.
2. Place the wetted Whatman® 3MM
3. paper on the gel surface and smooth to remove any airbubbles.
4. Overlay the paper with six layers of absorbent paper
toweling.
5. Place a glass plate on the paper towels. Weight it with a
1 kg-2 kg weight.
6. Leave in place for 20 minutes to overnight.
7. Remove the weight, glass plate and paper towels.
8. Re-wet the blotting paper to allow easy removal from thegel surface, and remove it.
9. Wash the gel for 5 minutes with deionized water.
10. Dry the gel in a 50°C to 55°C forced hot air oven for 30
minutes, overnight is fine. Gels may also be left to dry
overnight at room temperature.

Focusing Samples
Apply power to the gel (initial settings):
Power Limit Voltage Limit
Focusing Time25 W
1,000 to 2,000 V 60 to 90 minutes
Monitor the separation by:•Observing the separation of the visible marker proteins•
A drop in current to a low, steady state (1 mA-5 mA)
When focusing is complete, turn off power, discard the wicks
and place the gel in fixative.

Fixing, Staining and Destaining
Either Coomassie® Brilliant Blue or Crowle’s Double Stain canbe used to stain proteins in IsoGel® Agarose IEF Plates.
Coomassie® Brilliant Blue Stain will give the highest sensitivity.
Crowle’s Double Stain will give the clearest background, andslightly lower sensitivity. Individual proteins may stain more
intensely with one or the other stain.

Gel Staining Crowle’s Stain
1.

Preparation of Fixative for either Coomassie® orCrowle’s Stain

2.

Fixative Solution•180 ml methanol•30.0 g trichloroacetic acid•18.0 g sulfosalicylic acid•
Fill to 500 ml with deionized water

2Float the gel in the stain, gel side down, for 15 to 30
minutes. Floating gel side down in Crowle’s Stain
prevents particles in the stain solution from clinging to thegel.
Remove the gel from the stain, and gently rinse withrunning tap water until excess stain is removed. Gels may
be rinsed by placing in a 1500 ml beaker under a gentle
stream of tap water.

Gel Staining Coomassie® Stain
1.
2.

Stain gel 15 minutes in Coomassie® Stain with gentle
agitation.
Rinse gel in destaining solution 15-30 minutes with gentle
agitation.

Gel Drying And Preservation
Place in a 50°C to 55°C hot air oven for 15 minutes.
OR:
Dry at room temperature overnight.
NOTE: Dried gels can be stored indefinitely.

Ordering Information
Catalog No.
56015
56018
56014
56010

Description

Size

IsoGel® Agarose IEF
6 units
Plates-pH range 3-10
IsoGel® Agarose IEF
6 units
Plates-pH range 6-10.5
IsoGel® IEF Plate
Sufficient forAccessory Pack
6 plates
IsoGel® Agarose IEF
100 each
Plate Accessory Bulk Pack
Contains 125 mm wicks and blotting paper

For Laboratory Use.

Related Products
IsoGel® Agarose
GelBond® FilmPAGEr® Gold Precast Gels

For more information contact Technical Service at(800) 521-0390 or visit our website atwww.Lonza.com

GelBond is a trademark of FMC Corporation,Triton is atrademark of Union Carbide Chemicals & Plastics TechnologyCorp., Whatman is a trademark of Whatman Int’l Ltd.,
Rubbermaid and Servin’ Saver are trademarks of Rubbermaid,Inc., Tween is a trademark of ICI United States, Inc.,
Zwittergent is a trademark of Calbiochem-Novabiochem Corp.,
Nonidet is a trademark of Shell Int’l Petroleum Co., Coomassie
is a trademark of ICI, Lourdes. All other trademarks herein
are marks of the Lonza Group or its affiliates.
Copyright© 2007 Lonza Rockland, Inc.
All rights reserved.

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