1.seed cells for 24:00:00 in a 10 cm dish at a density of 6 x 106 cells per plate
2.wash cells with 1X PBS for 2 times
3.scrape cells of the dish and harvest in 1 ml pf PBS + PI buffer
4.1000 x g, 00:15:00
5.resuspend cells in 500 µl PBS + PI buffer
6.sonicate 2 times at 30Hz for 15 ON-OFF intervals of 10 s each (1 s pulse on/off).
7.340000 x g, 4°C, 00:15:00
collect supernatant as cytosolic fraction
8.was pellet 3 times with PBS + PI buffer
9.340000 x g, 4°C, 00:15:00
10.disolve pellet in 500 µL 1x PBS +PI+ 1% Trtiton
11.340000 rpm, 4°C, 00:15:00
collect supernatant as membranous fraction
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