Blue-Native Electrophoresis to Study the OXPHOS Complexes

Abstract

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique optimized for the analysis of the five components of the mitochondrial oxidative phosphorylation (OXPHOS) system. BN-PAGE is based on the preservation of the interactions between the individual subunits within the integral complexes. To achieve this, the complexes are extracted from the mitochondrial inner membrane using mild detergents and separated by electrophoresis in the absence of denaturing agents. The electrophoretic procedures can then be combined with a variety of downstream detection techniques. Since its development in the 1990s, BN-PAGE has been applied in the study of mitochondria from all kinds of organisms and extensive amounts of data have been produced using this technique, being key for the understanding of many aspects of OXPHOS physiopathology.

Keywords: Blue-native gel electrophoresis; First-dimension BN-PAGE; In gel activity assays; Mitochondrial complexes I, II, III, IV, and V; Oxidative phosphorylation system; Second-dimension BN-PAGE.