Acetic Acid-Urea Polyacrylamide Gel Electrophoresis of Basic Proteins

Abstract

Panyim and Chalkley described in 1969 a continuous acetic acid-urea (AU) gel system that could separate very similar basic proteins based on differences in size and effective charge (1). For instance, unmodified histone H4 can be separated from its monoacetylated or monophosphorylated forms (2). At the acidic pH 3 of this gel system, basic proteins with a high isoelectric point will clearly have a net positive charge that will be the major determinant of electrophoretic mobility. If a single of these positive charges is removed, for example, by in vivo acetylation of one of the positively charged ε-amino lysine side chain residues in the small histone H4 protein (102 residues), a significant decrease in effective gel mobility is observed. Similarly, addition of a phosphate moiety decreases the net positive charge of the protein during gel electrophoresis by one. Separation between similarly sized and charged proteins, for example, the partially acetylated H2A, H2B, and H3 histones of most organisms, can typically be achieved only by inclusion of a nonionic detergent such as Triton X-100 (see Chapter 17).