Microsomal membrane isolation from cell culture

harvest cells by detachment with TrypLE Express (Gibco, 12604-021)

pellet cells by centrifugation (5 min, 400 g_avg, 4°C)

wash cell pellet in phosphate buffered saline (PBS, Sigma, D8537-500ml)

pellet cells by centrifugation (5 min, 400 g_avg, 4°C)

resuspend pellet in RIPA lysis buffer (Thermo Scientific, 89900) supplemented with protease inhibitor (Sigmafast, Sigma, S8830-20TAB)

incubate 30min on ice

centrifuge lysate (10min, 100 g_avg, 4°C) to pellet and remove nuclear fraction

collect supernatant to continue isolation

centrifuge supernatant from previous step (10min, 15 000 g_avg, 4°C) to pellet the mitochondrial/lysosomal fraction

collect supernatant to continue isolation (pelleted mitochondrial/lysosomal fraction can be stored for future analysis)

centrifuge supernatant from previous step (35 min, 200 000 g_avg, 4°C) to pellet the microsomal fraction

resuspend pellet in sucrose (0.25 M)

for Western blotting prepare samples with 20µg protein with 4x LDS loading buffer (NuPAGE, invitrogen, 2399420)