Isoelectric Focusing of Proteins in Ultra-Thin Polyacrylamide Gels

Abstract

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 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, 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 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 charge.