34] Protein analysis using high-resolution two-dimensional polyacrylamide gel electrophoresis

Abstract

This chapter describes the use of high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein analysis. The use of 2D-PAGE has become increasingly popular during the past decade. 2D-PAGE allows the resolution of a complex protein mixture into more discrete components than 1D-PAGE because it separates on the basis of protein charge in addition to molecular weight. The most common uses of 2D-PAGE are the analysis of complex mixtures of proteins and the analysis of the posttranslational modification of proteins. 2D-PAGE can also provide valuable information about the molecular properties of proteins, including an estimate of the relative isoelectric points (p I ) and molecular weights of proteins. The preparation of samples for 2D-PAGE analysis is the most critical step in guaranteeing excellent reproducible results. If standard electrophoresis chambers are used, electrophoresis is carried out by placing the electrode buffer in the upper and lower chambers. The slab gels are then placed into these chambers, taking care to avoid air bubbles being trapped at the bottom of the slab acrylamide gel.