Blue Native (BN)-polyacrylamide gel electrophoresis (PAGE)

For BN-PAGE we used protocol as explained by Jha et al.⁶⁷. Mitochondrial protein extracts prepared in 1X Native PAGE™ Sample Buffer and detergents were subsequently run on a 4–16% Blue Native polyacrylamide gradient gel following the manufacturer’s protocol (Invitrogen, Native PAGE Gel system). Immediately prior to electrophoresis, NativePAGE™ 5% G-250 Sample Additive was added to the samples and the NativeMark™ Unstained Protein Standard (Invitrogen, LC0725) was used as the molecular weight markers. Two types of NativePAGE™ Running Buffers are used for native gel electrophoresis with NativePAGE™ Novex® Bis–Tris Gels. NativePAGE™ 20X Running Buffer (BN2001) and the NativePAGE™ 20X Cathode Buffer Additive (BN2002), contains 0.4% Coomassie G-250 (added to NativePAGE™ Running Buffer to generate the Cathode Buffer) which were purchased and prepared according to the manufactures protocol. The dark cathode buffer (Dark Blue Cathode Buffer contains 0.02% G-250) was used for the Blue native gel. The manufacturer’s instructions for performing electrophoresis using the Invitrogen Mini-Gel Tank (Invitrogen, A25977) was followed with the gel being run in a cold room at 4 °C. The gel was run first at 150 V for 60 min, then at 250 V for the remainder of the run (30–90 min). After electrophoresis was completed, a fast Coomassie G-20 staining was performed by first placing the gel in 100 ml Fix solution (40% methanol, 10% acetic acid) and microwaving on high (950–1100 watts) for 45 s. The gel was then placed on an orbital shaker for 15 min at room temperature. Finally, to destain the gel, we added 100 ml destain solution (8% acetic acid) and microwaved on high (950–1100 watts) 45 s. The final round of shaking was done on an orbital shaker at room temperature until the desired background was obtained. The gel was then washed with water and photographed using a White Light Transilluminator FB-WLT-417 (Fisher Scientific).