For trypsin digestion, 50 μg of intact mitochondria were taken in 50 μl of SEM buffer and treated with 1 μg, 2 μg, and 5 μg (final concentration) of trypsin (Amresco, 0785) on ice for 15 min. Protease treatment was stopped by adding 5 μg, 10 μg, and 25 μg (final concentration) soybean trypsin inhibitor (Amresco, K213), respectively, and incubated on ice for 15 min. Mitochondria were pelleted down at 14,000g for 10 min at 4 °C.
For high-salt treatment, 50 μg mitochondria in 50 μl of SEM buffer were incubated with 400 mM KCl on ice for 10 min. Supernatant and pellet were separated by centrifugation at 14,000 rpm for 15 min. For high pH treatment, mitochondria were incubated with 200 mM Na2CO3 (pH 11.5) for 15 min followed by centrifugation to separate the pellet and supernatant.
For high-salt treatment, 50 μg mitochondria in 50 μl of SEM buffer were incubated with 400 mM KCl on ice for 10 min. Supernatant and pellet were separated by centrifugation at 14,000 rpm for 15 min. For high pH treatment, mitochondria were incubated with 200 mM Na2CO3 (pH 11.5) for 15 min followed by centrifugation to separate the pellet and supernatant.