Bio-Rad Model 111 Mini IEF Cell - Manual (Page 9)

2.3 Choice of Support Matrix
The electrofocusing process must be stabilized against convection and, to a lesser extent,
diffusion, by a support matrix. This can be anything from a liquid column stabilized by a sucrose
density gradient to a gel cast from agarose or polyacrylamide. The principal criteria for a good
support matrix are that it should be relatively non-sieving so that molecular size is not a factor in
protein mobility, and that it must be free of charged groups which would give rise to internal fluid
flow and distortion of the pH gradient. A complete discussion of electrofocusing matrices is given
in Reference 1.
For analytical work, both agarose and polyacrylamide gels provide good supports for
electrofocusing. Agarose has the advantage of very large pore structures (as large as 500 nm),
making it an ideal non-sieving medium; however, it suffers from varying degrees of residual
negative charge from sulfate groups. For this reason, only agarose proven for electrofocusing
applications should be used (Bio-Rad’s Zero -Mr Agarose, catalog number 162-0022). Agarose
concentrations may vary between 0.5 and 1.25%. Proteins as large as 50 x 10
6
daltons have been
successfully electrofocused in agarose.
Section 3
Polyacrylamide Gel Isoelectric Focusing
3.1 Considerations in Matrix Preparation
Because they are prepared from monomers, polyacrylamide gels can be tailored to meet
particular separations requirements. The most common gel composition for horizontal
electrofocusing is T = 5%, C = 3%, where:
g acrylamide + g crosslinker
total solution volume in ml
g crosslinker
g acrylamide + g crosslinker
This formulation will give a suitable non-sieving gel for proteins up to 10 6 daltons, that is still
rigid enough to handle conveniently. A slightly stronger gel of T=5%, C = 4% may be used for
protein samples under 200,000 daltons.
The choice of a catalyst is extremely important in electrofocusing, since any residual ions will
affect the final attainable voltage, and can lead to overheating and gross distortions in the gel. For
this reason a three-phase catalyst system of ammonium persulfate, riboflavin-5'-phosphate, and
TEMED is recommended. This system, catalyzed by light, will give reproducible polymerization
with a minimum of ionic contamination.
The formation of polyacrylamide gels has been extensively studied, and a detailed discussion
of practical considerations is available in Bio-Rad’s bulletin 1156.
5
x 100
%C = x 100
%T =