Two-Dimensional Polyacrylamide Gel Electrophoresis for the Separation of Proteins for Chemical Characterization

Abstract

Two-dimensional polyacrylamide gel electrophoresis (2-DE) based on the method of O’Farrell (1) has an almost unrivaled capacity for the separation of complex mixtures of proteins. The standard method of 2-DE can routinely separate up to 2000 proteins from whole cell and tissue homogenates, and using large format gels separations of up to 10,000 proteins have been described (2). Until recently, 2-DE was used primarily as an analytical tool for the characterization of proteins by their charge (pI), size (Mr) and relative abundance. Sophisticated computer software has been developed for the quantitative and qualitative analysis of complex 2-DE profiles and to allow the comparative analysis of patterns of protein expression in a variety of biological systems (3). However, a major obstacle to the exploitation of 2-DE for the development of comprehensive protein databases has been that the method provides no direct clues as to the identity or function of the separated components. This problem has now been largely overcome with the development of a battery of highly sensitive techniques of microchemical characterization, including N-terminal and internal protein microsequencing, amino acid compositional analysis, peptide mass profiling, and mass spectrometry (4,5). This has resulted in 2-DE often being the method of choice for the micropreparative purification of proteins for subsequent chemical analysis. These chemical analysis methods are described in detail in the relevant chapters in the present volume.