Lonza Rockland, Inc.
www.lonza.com
biotechserv@lonza.com
Tech Service: 800-521-0390
Customer Service: 800-638-8174
Document # 18165-1007-06
Rockland, ME 04841 USA

Isoelectric Focusing with IsoGel® Agarose IEF Plates, pH 7-11
Introduction

Circulation BathIsoGel® Agarose IEF Plates, pH 7-11 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.

Set the temperature on the circulator bath so that the platen
temperature reaches 10°C. Chilling must occur enough inadvance to ensure platen temperature is 10°C.

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

Storage•Removing IsoGel® Plate From CassetteStore 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 PlateAccessory Pack catalog # 56010 and 56014)•Horizontal Isoelectric Focusing Chamber. The Multiphor®
II Electrophoresis Unit from Amersham Pharmacia
BioTech is ideal for use with IsoGel® Agarose IEF Plates.
•Constant Power Supply•
Refrigerated Circulator Bath•Forced Air Oven set at 50°C to 55°C•
pI Marker Proteins•
Micropipettor and Pipette tips

NOTE: A frosted or crystalline background on a gel when first
opened may indicate the product was frozen. If this occurs
contact Technical Service for assistance.
1.
2.

3.

Gel Placement

Electrolyte Solutions Required••

Anolyte:Catholyte:

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

1.

0.01 M Glacial Acetic Acid
Cathode Fluid 10,
Cresent Chemicals
catalog #42986.03

2.

Preparing Protein Samples

3.

For protein samples containing salts, dialyze the protein
sample against one of the following:•Deionized water•
1% glycine•
0.05 M to 0.1 M ammonium bicarbonate.

4.

For samples that are hydrophobic or poorly soluble, add either
nonionic or zwitterionic detergent to the sample at a finalconcentration of 0.05% to 1.0%
•Nonionic detergents•Triton® X-100•
Nonidet® (NP-40)•Tween® 80•
Zwitterionic detergents•CHAPS•
Zwittergent® 3-14

5.

Wipe any condensation off the cooling platen. The surfaceof the electrophoresis platform should be between 10°C
to 15°C.
Spread a small volume (100 μl) of deionized water onto
the center of the cooling platen of the IEF chamber. This
will hold the gel to the platen by capillary action.
Lower the gel on the wetted area.
NOTE: Avoid trapping air between the GelBond® Film andplaten. If bubbles form, use the edge of a scalpel blade or
thin spatula to gently lift the edge of the gel, then lay thegel back down on the platen to remove the bubble.
Wipe or blot excess fluid from the area around the gel,right up to the edges of the gel.
Briefly blot the surface of the gel with a sheet of blotting
paper.

Electrode Wick Application
1.

2.

1

Cut wicks to 9 cm in length. This length is ideal because it
allows 0.5 cm space between the edges of the gel andeach end of the wick.
Completely immerse a wick in the catholyte solution(Cathode Fluid 10). This is the cathodal wick.

3.

4.

5.
6.
7.
8.

Remove excess fluid from the wick by placing it briefly on
blotting paper, then gently blot the top surface of the wickwith blotting paper.
Repeat steps 1 and 2 with another wick using the anolyte
solution (0.01 M gacial acetic acid). This is the anodalwick.
Place the cathodal wick at the (-) electrode contact of thegel, centered on the gel.
Place the anodal wick at the (+) electrode contact of thegel, centered on the gel.
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 gel surface.

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 water
Coomassie® Destaining Solution
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 ml glacial acetic acid30.0 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. Positioning of sample application may be
adjusted depending on the pI of the proteins. We
recommend loading samples 3 cm from anode 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 the wicks 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 waterGel Fixation
1.
2.
3.

Place the gel in fixative for 20 to 30 minutes.
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.
2.
3.
4.

Focusing Samples

5.
6.

Apply power to the gel (initial settings):
Power Limit Voltage Limit Focusing Time
25 W
1000 V
60 to 90 minutes

7.
Monitor the separation by:•Observing the separation of 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.

8.

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

Gel Staining Crowle’s Stain
1.

Fixing, Staining and Destaining
Background2.

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.

Float 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 the gel.
Remove the gel from the Crowle’s Stain, and gently rinse
with running tap water until excess stain is removed. Gels
may be rinsed by placing in a 1,500 ml beaker under agentle stream of tap water.

Gel Staining Coomassie® Stain
1.

Preparation of Fixative for Either Coomassie® or
Crowle’s Stain

2.

Fixative Solution
180 ml methanol
30.0 g trichloroacetic acid18.0 g sulfosalicylic acid (e.g. Sigma Catalog S-2130)
Fill to 500 ml with deionized water

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.

2

NOTE: Dried gels can be stored indefinitely.

Ordering Information
Catalog No.
56024

Description

IsoGel® Agarose IEFPlates pH 7-11

Size
6 plates

Related Products

IsoGel® Agarose IEF Plate pH range 3-10
IsoGel® Agarose IEF Plate pH range 6-10.5®
IsoGel Agarose
IsoGel® IEF Plate Accessory Pack®
GelBond Film®
PAGEr Gold Precast Gels

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

For Laboratory Use.
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., Tween isa trademark of ICI United States, Inc., Zwittergent is atrademark of Calbiochem-Novabiochem Corp., Nonidet is atrademark of Shell Int’l Petroleum Co., Coomassie is atrademark of ICI, Lourdes. Multiphor is a trademark ofPharmacia Biotech AB Corporation. All other trademarks
herein are marks of the Lonza Group or its affiliates.
© 2007 Lonza Rockland, Inc.
All rights reserved.

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