4 Generation of pH gradients

Abstract

Isoelectric focusing (IEF) is the electrophoretic separation of proteins in a pH gradient. The pH gradient is critical to the technique and the nature of the pH gradient largely determines the quality and usefulness of the separation. The different IEF methods that have been developed over the years are largely distinguished from one another in terms of the means by which the pH gradient is formed. The pH gradient should fulfill a number of requirements to be useful for IEF separations. The pH gradient should be reproducible so that the results of separations can be compared with one another. The pH gradient used for IEF can either be “artificial” or “natural.” A “natural” pH gradient arises solely through the action of electrical current passing through a solution that is homogeneous prior to application of the current. The development of synthetic carrier ampholytes was the first breakthrough that allowed the formation of pH gradients fulfilling everything required to be used routinely for highly resolving IEF. The chapter discusses the practical aspects of carrier ampholyte-generated pH gradients. Of all the pH gradient generation schemes developed in response to the disadvantages of carrier ampholytes, the most successful and widely applied has been the so-called immobilized pH gradient (IPG). Recipes for acrylamido buffer mixtures for the generation of immobilized pH gradients are available from several sources.