Resolving mitochondrial protein complexes using nongradient blue native polyacrylamide gel electrophoresis

Blue native polyacrylamide gel electrophoresis (BN–PAGE) is a powerful technique for separation and proteomics analysis of high-molecular-weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this article, we present an alternative BN–PAGE method that uses highly porous, nongradient polyacrylamide gels for separation of rat brain mitochondrial protein complexes. Results demonstrate that this method not only resolves mitochondrial complexes I to V, allowing subsequent analysis by in-gel activity staining and mass spectrometry peptide sequencing, but also identifies Hsp60 polymers and dihydrolipoamide dehydrogenase (DLDH). Moreover, with this new method, it is shown for the first time that complex I and DLDH can be detected simultaneously on a single gel strip by in-gel activity staining. Overall, the method provides a simplified, nongradient gel electrophoretic approach that should be useful in functional proteomics studies.