Drp1 was expressed in One Shot BL21 Star (DE3) Escherichia coli (C6010-03; Life Technologies, Carlsbad, CA) by isopropyl-β-d -thiogalactoside induction at 20°C for 16 h. Cell pellets were resuspended in lysis buffer (100 mM Tris-Cl, pH 8.0, 500 mM NaCl, 1 mM dithiothreitol [DTT], 1 mM EDTA, 2 μg/ml leupeptin, 10 μg/ml aprotinin, 2 μg/ml pepstatin A, 2 mM benzamidine, 1 μg/ml calpain inhibitor I [ALLN], and 1 μg/ml calpeptin) and lysed using a high-pressure homogenizer (M-110L Microfluidizer Processor; Microfluidics, Newton, MA). The lysate was clarified by centrifugation at 40,000 rpm (type 45 Ti rotor; Beckman, Brea, CA) for 1 h at 4°C. Avidin (20 μg/ml; PI-21128; ThermoFisher Scientific, Waltham, MA) was added, and then the supernatant was loaded onto Strep-Tactin Superflow resin (2-1206-025; IBA, Göttingen, Germany) by gravity flow. The column was washed with 20-column volumes of lysis buffer without protease inhibitors. To elute Drp1, 0.01 mg/ml HRV3C protease in lysis buffer without protease inhibitors was added for 16 h at 4°C. The Strep-Tactin Superflow eluate was further purified by size exclusion chromatography on Superdex200 (GE Biosciences, Piscataway, NJ), spin concentrated, frozen in liquid nitrogen, and stored at −80°C.
Rabbit skeletal muscle actin was extracted from acetone powder as previously described (Spudich and Watt, 1971 (link)), and further purified by size exclusion chromatography on Superdex 75 (GE Biosciences). Actin was stored at 4°C in G-buffer (2 mM Tris, pH 8.0, 0.5 mM DTT, 0.2 mM ATP, 0.1 mM CaCl2, and 0.01% NaN3).
Rabbit skeletal muscle actin was extracted from acetone powder as previously described (Spudich and Watt, 1971 (link)), and further purified by size exclusion chromatography on Superdex 75 (GE Biosciences). Actin was stored at 4°C in G-buffer (2 mM Tris, pH 8.0, 0.5 mM DTT, 0.2 mM ATP, 0.1 mM CaCl2, and 0.01% NaN3).
