Detection of Mitochondrial Respiratory Chain Complexes by BN‐PAGE and Its Applications

ABSTRACT Blue native polyacrylamide gel electrophoresis (BN‐PAGE) is a core technique for separating and characterizing plant mitochondrial respiratory chain complexes. Under mild, nondenaturing conditions, Coomassie brilliant blue G‐250 binds to membrane protein complexes, imparting a negative charge while preserving native conformation and enzymatic activity. Under optimized conditions, this allows the simultaneous analysis of all five oxidative phosphorylation complexes (CI–CV) and their supercomplex assemblies on a single gel. In recent years, the combination of BN‐PAGE with immunoblotting and in‐gel activity staining has significantly expanded its utility in assessing complex assembly status, resolving subunit composition, and detecting enzymatic activities. This review systematically summarizes the technical principles, essential reagent components, and standardized protocols of BN‐PAGE and highlights its recent applications in plant developmental and fruit ripening studies, abiotic stress and postharvest disease resistance research, and mitochondrial RNA metabolism studies. The advantages and limitations of this technique compared with conventional methods are also addressed. This review aims to provide a comprehensive methodological reference for plant mitochondrial research and to promote the standardized application of BN‐PAGE in plant science.