BN-PAGE of Ustilago maydis Respiratory Complexes

Isolated mitochondria are valuable for the study of the respiratory chain, protein complex assembly, and the activities of the Krebs cycle enzymes, among others. Here, we present a protocol for the isolation of functional U. maydis mitochondria, which involves mechanically disrupting the cell wall with glass beads and performing two centrifugations. The first step is carried out at low speed to remove intact cells and cell debris, followed by a high-speed centrifugation to obtain mitochondria. For the separation of respiratory complexes and supercomplexes, we describe the solubilization of protein complexes from mitochondria with digitonin and their separation by blue native polyacrylamide gel electrophoresis (BN-PAGE), followed by the in-gel activity of respiratory complexes.