We recommend cell lysis with a mild detergent such as Triton X-100 or NP-40 when downstream applications like immunoprecipitation are to be used.
The protocol refers to lysis of adherent cells but can be easily adapted to cells growing in suspension.
1. Wash the cell plate with ice-cold PBS. Repeat 3 times.
2. Place the plate on ice and add ice-cold lysis buffer (50 mM Tris pH 7.6, 150 mM NaCl, 5 mM EDTA pH 8, 1% Triton X-100 and protease inhibitor cocktail) at a 5×106 cells/ml lysis buffer ratio.
3. Scrape the dish with a cell scraper and collect the lysate into a microtube.
4. Rock the sample in the microtube for 30 minutes at 4°C.
5. Centrifuge the sample at 14,000 rpm for 10 minutes at 4°C.
6. Carefully transfer the clear supernatant to a clean microtube. The sample can be stored at -20°C for several months or immediately mixed with sample buffer and loaded on an SDS/PAGE gel.
Note: Cell count can be established by dislodging the cells with trypsin from a parallel plate and counting them.