Gradient blue native gel electrophoresis (BN-PAGE)

A BN-PAGE system was assembled with gradient Mini-PROTEAN® TGX™ Precast Gels (4–15%) (BioRad). The inner compartment was filled with cathode buffer containing 50 mM Tricine (Sigma-Aldrich), 15 mM Bis Tris and 0.01% Brilliant Blue G. Twenty µL of each sample (containing 70 µg of protein) were loaded into each well. The outer compartment was filled with anode buffer containing 50 mM Bis Tris and the gel was run at 75 V. When the blue front reached one-third of the gel the cathode buffer was replaced with one devoid of brilliant blue G. Gel was then run at 150 V until the blue front reached the bottom of the gel, in the tank surrounded by ice. After removal from the cast the gel was incubated at 37 °C with 50 mL Complex I substrate, containing 1 M Tris-HCl, 4 mg of NADH and 10 mg of nitrotetrazolium blue chloride (in 200 µL EtOH), for 10 min with shaking. Gel images were developed using a VersaDocTM imaging system (Bio-Rad).