seed cells for 24h 0m 0s in a 10 cm dish at a density of 6 x 10⁶cells per plate

wash cells with 1X PBS for 2 times

scrape cells of the dish and harvest in 1 ml pf PBS + PI buffer

1000x g

resuspend cells in 500 µl PBS + PI buffer

sonicate 2 times at 30Hz for 15 ON-OFF intervals of 10 s each (1 s pulse on/off).

340000x g,4°C

collect supernatant as cytosolic fraction

was pellet 3 times with PBS + PI buffer

340000x g,4°C

disolve pellet in 500µL 1x PBS +PI+ 1% Trtiton

340000rpm,4°C

collect supernatant as membranous fraction