Transfect cells with PEI 2 days before in 10cm petri dish
wash cells PBS x2
add 3 ml Trypsin-EDTA
5 min RT
count cells
resuspend in 2x106 cells/ml in DMEM -FCS 37°C 5 min + 5mM DTT (final, fresh made) (50 µl DTT 0.1M)
wash cells 2x PBS 4°C
centrifuge 4°C pelletting cells
divide cells in different Eppendorf (1x106 cells/ml) in complete 10% FCS DMEM and put at the same time (few samples) in a 37°C or 24°C water bath
at different times add NEM (10 µl NEM 1M) and put in ice: 1=0, 2=2 min, 3=4 min, 4=8 min
wash 4°C PBS 1x
lysis of cells in RIPA 10mM NEM + cocktail protease inhibitors: 15 min ice - 10x106 cells/ml (tot 100 µl)
load 10µl on NR SDS-PAGE 12% MIDI