Isoelectric Focusing in Polyacrylamide Gel

Abstract

From many viewpoints, isoelectric focusing (IEF) represents a major advance in methodology for high-resolution separation of proteins and other amphoteric macromolecules. IEF is essentially an equilibrium method for segregating amphoteric molecules by electrophoresis in stable pH gradients according to their isoelectric points (pI). In only one or two experiments it is theoretically possible to display all components whose pI values differ by as little as 0.01 pH units. Such exquisite resolution by charge is not normally obtainable by other procedures such as electrophoresis or ion-exchange chromatography. Because of this built-in resolution, IEF is a more definitive technique for examining charge heterogeneity and is, therefore, a rigorous test of homogeneity. In addition, because substances are also concentrated as they are separated by I EF, the technique lends itself to both analytical and preparative purposes. Finally, IEF defines an important parameter, the isoelectric point (pI), which gives information not only about the composition and conformation of macromolecules but also allows a rational approach to further experimental manipulations.