Membrane Fractionation
Veerle Baekelandt
Published: 2021-08-05 DOI: 10.17504/protocols.io.bw5vpg66
Abstract
Membrane Fractionation (Ultracentrifugation)
Steps
1.
seed cells for 24h 0m 0s in a 10 cm dish at a density of 6 x 106cells 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.
1000x g
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.
340000x g,4°C
collect supernatant as cytosolic fraction
8.
was pellet 3 times with PBS + PI buffer
9.
340000x g,4°C
10.
disolve pellet in 500µL 1x PBS +PI+ 1% Trtiton
11.
340000rpm,4°C
collect supernatant as membranous fraction
