Two-dimensional blue native polyacrylamide gel electrophoresis

Abstract

Multiprotein complexes play crucial roles in nearly all cell biological processes. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a powerful method to study these complexes. It is a native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. It has a higher resolution than gel filtration or sucrose density ultracentrifugation and can be used for protein complexes from 10 kDa to 10 MDa. If a second-dimension SDS-PAGE is applied (two-dimensional BN/SDS-PAGE), the size, subunit composition, and relative abundance of the different multiprotein complexes can be studied. In recent years, there has been a large increase in the number of publications where BN-PAGE was used to study protein-protein interactions. Here, we give detailed protocols for the separation of multiprotein complexes by two-dimensional BN/SDS-PAGE and for a related technique to determine the stoichiometry of these complexes.