Lonza Rockland, Inc.
www.lonza.com
biotechserv@lonza.com
Tech Service: 800-521-0390
Customer Service: 800-638-8174
Document # 18164-1007-06
Rockland, ME 04841 USA

IsoGel® Agarose IEF Plates, pH 6-10.5
Nonionic detergents•Triton® X-100•
Nonidet® (NP-40)•Tween® 80

Introduction

IsoGel® Agarose IEF Plates, pH 6-10.5 are prepared bycustom blending IsoGel® Agarose with ampholytes. They form
a less restrictive matrix than polyacrylamide and are ideal forrapid focusing of high molecular weight proteins (>200 kDa).
They are particularly well suited for the separation and analysisof antibodies. IsoGel® Agarose is cast on GelBond® Film toprovide dimensional stability and ease of handling throughout
processing. Plate dimensions are 100 mm x 125 mm.

Zwitterionic detergents•CHAPS•
Zwittergent® 3-14

Circulation BathStorage

Set the temperature on the circulator bath so that the platen
temperature reaches 10°C-15°C. Chilling must occur enoughin advance to ensure platen temperature is 10°C.

Store plates at 4°C

Materials Required••
••

•
•••
•

Sample Applicator Mask
Electrode WicksBlotting Paper(The above three items are included in the IsoGel® IEF
Plate Accessory Pack, Catalog No. 56014,
100 wicks and 100 sheets blotting paper areCatalog No. 56010)
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 BathForced Air Oven set at 50°C to 55°C
pI Marker Proteins
Micropipettor and Pipette tips

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.

Removing IsoGel® Plate From Cassette
NOTE: Remove gel from refrigerator and use immediately.
Gels left at room temperature for extended periods may adhere
to the upper cassette when the cassette is opened.
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.

Electrolyte Solutions Required••

Anolyte: 0.05 M Glacial Acetic Acid OR 0.1 M Glutamicacid (Sigma G-5513)
Add 0.147 g to 10 ml dH2O, do not adjust pH
Catholyte: Cathode Fluid 10, Cresent Chemical
Catalog #42986.03

3.

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 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 one of the following:•Deionized water•
1% glycine•0.05 M to 0.1 M ammonium bicarbonate.

2.

3.

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%

1Wipe any condensation off the cooling platen. The surfaceof the electrophoresis platform should be between 10°C
and 15°C.
Spread a small volume (100 μl) of deionized water onto
the center of the cooling platen of the IEF chamber. Thiswill 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.

4.
5.

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 blottingpaper.

Preparation of Fixative for either Coomassie® or
Crowle’s Stain
Fixative Solution
180 mlMethanol
30.0 gTrichloroacetic acid18.0 gSulfosalicylic acid (e.g., Sigma catalog S-2130)
Fill to 500 ml with deionized waterElectrode Wick Application
1.

2.
3.

4.
5.
6.
7.
8.

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.
Remove excess fluid from the wick by placing it briefly on
blotting paper, and then gently blot the top surface of thewick with blotting paper.
Repeat steps 1 and 2 with another wick using the anolyte
solution (0.1 M Glutamic acid). This is the anodal wick.
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 mlEthanol
90 ml
Glacial acetic acid
Fill to 1 L with deionized water

Coomassie® Destaining Solution
250 mlEthanol
90 ml
Glacial acetic acid
Fill to 1 L with deionized 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
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 beadjusted 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 water

Gel Fixation
1.

2.
3.

Gel Drying
1.
2.
3.

Focusing Samples
1.

Apply power to the gel as follows (initial settings):
Power Limit
25 W

2.

3.

Voltage Limit
1000 V

4.

Focusing Time
60 to 90 minutes

5.
6.

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 thewicks and place the gel in fixative.

7.
8.

Fixing, Staining and Destaining
Background

Place the gel in fixative for 20 to 30 minutes.
®
®
(Rubbermaid Servin Saver Rectangle #3865 or®
®
Rubbermaid Servin Saver 2 or 5 cup square containers
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.
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. Rewet the blotting paper to allow easy removal from the gel
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.

Either Coomassie Brilliant Blue or Crowle’s Double Stain can®
be 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.

2.

2

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 1500 ml beaker under agentle 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.

For Laboratory Use.
For more information contact Technical Service at(800) 521-0390 or visit our website atwww.Lonza.com
GelBond is a trademark of FMC Coproration, Triton is a trademark ofUnion Carbide Chemicals & Plastics Technology Corp., Whatman is atrademark of Whatman Int’l Ltd., Tween is a trademark of ICI UnitedStates, 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. Rubbermaid and Servin’ Saver aretrademarks of Rubbermaid, Inc. Multiphor is a trademark ofAmersham BioSciences. All other trademarks herein are marks of theLonza Group or its affiliates.
© 2007 Lonza Rockland, Inc.
All rights reserved.

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