Protein Protocols Protein Protocols

Ultra-Thin Polyacrylamide Gels 125
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Isoelectric Focusing of Proteins
in Ultra-Thin Polyacrylamide Gels
John M. Walker
1. Introduction
Isoelectric focusing (IEF) is an electrophoretic method for the separation of proteins,
according to their isoelectric points (pI), in a stabilized pH gradient. The method
involves casting a layer of support media (usually a polyacrylamide gel but agarose can
also be used) containing a mixture of carrier ampholytes (low-mol-wt synthetic
polyamino-polycarboxylic acids). When using a polyacrylamide gel, a low percentage
gel (~4%) is used since this has a large pore size, which thus allows proteins to move
freely under the applied electrical field without hindrance. When an electric field is
applied across such a gel, the carrier ampholytes arrange themselves in order of
increasing pI from the anode to the cathode. Each carrier ampholyte maintains a local
pH corresponding to its pI and thus a uniform pH gradient is created across the gel. If a
protein sample is applied to the surface of the gel, where it will diffuse into the gel, it
will also migrate under the influence of the electric field until it reaches the region of
the gradient where the pH corresponds to its isoelectric point. At this pH, the protein
will have no net charge and will therefore become stationary at this point. Should the
protein diffuse slightly toward the anode from this point, it will gain a weak positive
charge and migrate back towards the cathode, to its position of zero charge. Similarly
diffusion toward the cathode results in a weak negative charge that will direct the protein
back to the same position. The protein is therefore trapped or “focused” at the pH
value where it has zero overall charge. Proteins are therefore separated according to
their charge, and not size as with SDS gel electrophoresis. In practice the protein
samples are loaded onto the gel before the pH gradient is formed. When a voltage
difference is applied, protein migration and pH gradient formation occur simultaneously.
Traditionally, 1–2 mm thick isoelectric focusing gels have been used by research
workers, but the relatively high cost of ampholytes makes this a fairly expensive procedure
if a number of gels are to be run. However, the introduction of thin-layer isoelectric
focusing (where gels of only 0.15 mm thickness are prepared, using a layer of
electrical insulation tape as the “spacer” between the gel plate) has considerably reduced
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
125