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 (p
I
), size (
Mr
), 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 .