Native page 3 12 bis tris

75 μg of mitochondria were pelleted by centrifugation and resuspended in BN-lysis buffer (Wittig et al., 2006 (link)). For analysis of F₁F_O-ATP synthase or MICOS complex 1 % (w/v) or 3 % (w/v) digitonin were used. Cleared lysates were supplemented with Native PAGE 5% G-250 Sample Additive and subjected to BN-PAGE (Native PAGE 3–12% Bis-Tris; Life Technologies, Carlsbad, CA, USA). After blotting on PVDF membranes (Roth, Karlsruhe, Germany) immuno-decoration using the indicated antibodies was performed.

Mitochondria (150 µg protein) were pelleted by centrifugation (12 min at 17,600×g, 4°C) and resuspended in 15 µl BN-lysis buffer (50 mM NaCl, 50 mM imidazole, 2 mM 6-aminohexanoic acid, 1 mM EDTA, 1 mM PMSF, 3% digitonin, pH 7.0) (Wittig et al., 2006 (link)). After incubation for 15 min on ice, a clarifying spin was performed (20 min at 15,000 × g, 4°C). To the cleared lysates 1.5 µl Native PAGE 5% G-250 Sample Additive (Life Technologies, Carlsbad, CA, USA) was added and the samples were subjected to BN-PAGE (Native PAGE 3–12% Bis-Tris; Life Technologies). After blotting on PVDF membranes (Roth, Karlsruhe, Germany) immunoblotting was performed.