Systematic Monitoring of Protein Complex Composition and Abundance by Blue-Native PAGE

Abstract

INTRODUCTIONMany polypeptides do not perform their functions as single autonomous units in vivo. Instead, multiple polypeptides associate to form higher molecular mass structures. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) allows a range of the major protein complexes involved in such protein-protein interactions to be visualized simultaneously and in a single experiment. When combined with a second dimension of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the BN/SDS-PAGE procedure can resolve the complexes according to their molecular weight, as well as the subunits within each complex, according to the molecular weights of the subunits. Similarly, used in conjunction with differential in-gel electrophoresis (DIGE), it can accurately quantify changes in protein complex abundance or subunit composition between different samples, or between different complexes within the same sample. The following basic protocol describes sample preparation and gel casting for the first (BN-PAGE) and second (SDS-PAGE) dimensions. Variants are presented with and without DIGE labeling, along with the additional steps required for the fluorescence DIGE technique.