Mitochondrial Isolation and Blue Native Electrophoresis

Mitochondria were isolated from mouse tissue using differential centrifugation as previously described (31 (link)). For blue native electrophoresis, 100 µg mitochondria were solubilized in solubilization buffer: 1% (w/v) digitonin (Calbiochem), 20 mM Tris, pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% (v/v) glycerol. Following 15 min of incubation on ice, non-solubilized material was removed by centrifugation and the supernatant was mixed with loading dye (5% (w/v) Coomassie Brilliant Blue G-250 (Serva), 100 mM Tris pH 7, 500 mM 6-aminocaproic acid). Samples were resolved on 4–10% (w/v) acrylamide gradient BN-PAGE gels (50 (link)). BN gels were further subjected to western blot analysis or Coomassie Brilliant Blue R staining as indicated.