Detection of Total Proteins on Western Blots of 2-D Polyacrylamide Gels

The ability of two-dimensional electrophoresis (2-DE), based on the original method developed more than 20 years ago (1 ), to separate simultaneously up to several thousand proteins using large-format gels (2 ) has made it the method of choice for the analysis of protein expression in complex biological systems, such as whole cells, tissues, and organisms. Initially, 2-DE only yielded information on the charge (pI ), size (M _r ), and relative abundance of the separated proteins. However, in recent years, a variety of methods have been developed that make it possible to identify and characterize proteins separated by 2-DE. Many of these methods depend on the technique of Western electroblotting in which proteins separated by 2-DE are transferred (“blotted”) by the application of an electric field perpendicular to the plane of the gel onto the surface of an inert membrane, such as nitrocellulose (3 ). Methods for electroblotting of protein from 2-DE gels are described in Chapter 35 .