Blue native polyacrylamide gel electrophoresis (BN-PAGE) is one of the useful methods to isolate protein complexes including membrane proteins under native conditions. In BN-PAGE, Coomassie Brilliant Blue G-250 binds to proteins and provides a negative charge for the electrophoretic separation without denaturing at neutral pH, allowing the analysis of molecular mass, oligomeric state, and composition of native protein complexes. BN-PAGE is widely applied to the characterization of soluble protein complexes as well as isolation of membrane protein complexes from biological membranes such as the complexes I-V of the mitochondrial respiratory chain and subcomplexes of the mitochondrial protein import machinery. BN-PAGE has also been introduced in the field of peroxisome research, for example, analysis of translocation machinery for peroxisomal matrix proteins embedded in the peroxisomal membrane. Here, we describe a basic protocol of BN-PAGE and its application to the study of peroxisome biogenesis.
Keywords: Blue Native PAGE; PEX genes; Peroxins; Peroxisome; Peroxisome biogenesis disorders; Peroxisome-deficient mutant cells; Protein complex; Protein import.