Bio-Rad Model 111 Mini IEF Cell - Manual (Page 18)

Section 6
Agarose Gel Electrofocusing
6.1 Introduction
Agarose isoelectric focusing separates large proteins and antibodies that cannot be readily
characterized on polyacrylamide IEF gels due to polyacrylamide’s smaller pore sizes. Molecules
greater than 200,000 daltons can easily be separated on a 1% agarose gel.
The agarose gels are formed within minutes by simply heating the agarose mixture and
pouring it into the casting tray much the same way as casting acrylamide gels. Sorbitol and
glycerol are incorporated into the agarose gel to increase gel viscosity and to counteract
electroendosmosis (EEO), a major cause of sample smearing. EEO is the cathodic flow of water
in the neutral and alkaline parts of the gel caused by the low concentration of fixed charge
carboxyl groups on the gel matrix. These carboxyl groups are not charged at pH 3.5 or lower, but
acquire their full charges at pH 5.5 and higher. As a consequence, gel shrinkage occurs at the pK
of the carboxyl groups, resulting in “flooding” of water and solutes at the alkaline portion of the
gel. EEO decreases when gel viscosity increases.
The procedure for agarose isoelectric focusing is simple, consisting of the following seven
steps. Each of these steps is described in detail in Sections 6.3 through 6.6.
1. Cast the agarose gel.
2. Dehydrate the gel to remove excess liquid.
3. Focus the gel for 90 minutes.
4. Immerse the gel in fixative solution.
5. Immerse the gel in ethanol to remove background.
6. Immerse the gel in stain.
7. Destain the gel.
6.2 Preparing Agarose Gels
Agarose IEF gel contains:
1% agarose, zero-Mr
2% ampholytes
5% sorbitol
10% glycerol
1. Add 0.5 grams of Bio-Rad’s Zero-Mr Agarose and 2.5 grams of sorbitol to 20 ml of 25%
glycerol and 10 ml of distilled water. Place a stir bar into the flask and immerse the flask in a
beaker of water. Heat the water to boiling (100 °C) and stir to dissolve the components (30
minutes).
2. Place the casting tray into a prewarmed 55 °C oven and allow it to equilibrate. The tray should
remain in the oven until just prior to pouring the gels.
3. Turn off heat and add ampholytes while stirring the agarose mixture (2.5 ml of 40%
ampholytes or 5 ml of 20% ampholytes). Add more hot (100 °C) distilled water for a final
volume of 50 ml.
4. Allow solution to cool to about 55 °C before pouring agarose. A hot water bath set to 55 °C is
optimal.
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