NativePAGE (BN-PAGE) and native immunoblots

The NativePAGE Novex bis-tris gel system (Thermo Fisher Scientific) was used to analyze native membrane protein complexes and native mitochondrial matrix complexes, with the following modifications: Only the light blue cathode buffer was used; 20 μg of membrane protein extracts were loaded per well; the electrophoresis was performed at 150 V for 1 hour and 250 V for 2 hours. For the native immunoblot, polyvinylidene difluoride was used as the blotting membrane. The transfer was performed at 25 V for 4 hours at 4°C. After transfer, the membranes were washed with 8% acetic acid for 20 min to fix the proteins and then rinsed with water before air-drying. The dried membranes were washed five to six times with methanol (to remove residual Coomassie Blue G-250), rinsed with water, and then blocked for 2 hours at room temperature in 5% milk, before incubating with the desired antibodies diluted in 2.5% milk overnight at 4°C. To avoid strip and reprobing of the same membrane, which might allow detection of signals from the previous immunoblots, samples were loaded and run in replicates on adjacent wells of the same gel and probed independently with different antibodies.