Chapter 3 - Conventional Isoelectric Focusing and Immobilised pH Gradients

Abstract

This chapter describes conventional isoelectric focusing (IEF) and immobilized pH gradients (IPGs). It is generally believed that conventional IEF has been fully developed and has definitely reached a plateau. IPGs, though in their infancy, have been extensively developed, both in their analytical as well as preparative aspects. IPGs are based on the principle that the pH gradient, which exists prior to the IEF run itself, is copolymerized, and thus insolubilized, within the fibers of the polyacrylamide matrix. This is achieved by using as buffers a set of seven nonamphoteric, weak acids and bases, having the general chemical composition: CH2=CH–CO–NH–R, where R denotes either one of three different weak carboxyls, with pKs 3.6, 4.4, and 4.6, or one of four tertiary amino groups, with pKs 6.2, 7.0, 8.5, and 9.3. During gel polymerization, these buffering species are efficiently incorporated into the gel. Immobiline-based pH gradients can be cast in the same way as conventional polyacrylamide gradient gels by using a density gradient to stabilize the Immobiline concentration gradient, with the aid of a standard, two-vessel gradient mixer.