Chapter 13 Blue-native gels to isolate protein complexes from mitochondria

Abstract

Publisher Summary This chapter presents a new buffer system for blue-native polyacrylamide gel electrophoresis (BN–PAGE) that is employed for the isolation of protein complexes from mitochondria. Several suggestions for choice of specific detergent and of the appropriate detergent or protein ratio for preservation of native protein–protein interactions are discussed. The application range of BN–PAGE gradually expanded from the analysis of mitochondrial oxidative phosphorylation system (OXPHOS) complexes from bovine and human tissue (mitochondrial encephaolomyopathies) to the analysis of OXPHOS systems from plant mitochondria, yeast OXPHOS mutants, and chloroplasts. BN–PAGE has been applied with great success to the analysis of mitochondrial protein import complexes and of receptors in signal transduction. BN–PAGE is also a valuable technique for the identification of physiological protein–protein interactions: preprotein translocase complexes linked together by their substrates, the preproteins, yeast ATP synthase existing in a dimeric state, and respiratory chain complexes from yeast and mammalian mitochondria organized to form a network of supercomplexes. Moreover, the application of BN–PAGE will expand to the analysis of further receptors in the plasma membrane and in the membranes of cellular organelles, to the analysis of protein–chaperone interactions, and to the detection and analysis of antigen–antibody interactions. Protein–antibody interactions are stable under the conditions of BN–PAGE, as deduced from a shift of the apparent molecular mass of the protein antigen, and detection of immunoglobulin G (IgG) heavy and light chains in 2D sodium dodecyl sulfate (SDS)–PAGE at the position of the protein antigen.