Analysis of membrane protein complexes by blue native PAGE

Abstract

Blue native polyacryamide gel electrophoresis is a special case of native electrophoresis for high resolution separation of enzymatically active protein complexes from tissue homogenates and cell fractions. The method is powerful between 10 and 10,000 kDa. Also membrane protein complexes are separated well after solubilization of complexes with mild neutral detergents. The separation principle relies on binding of Coomassie blue G250 which provides negative charges to the surface of the protein. During migration to the anode, protein complexes are separated according to molecular mass and/or size and high resolution is obtained by the decreasing pore size of a polyacrylamide gradient gel. The principles of 2-dimensional blue native sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented here together with a practical step-by-step guide to performing the method in the laboratory.