The simplest method for preparing cell lysates is to lyse them directly with electrophoresis (Laemmli) sample buffer. With this method almost all cell proteins are released into the buffer and are readily available for separation on standard SDS/PAGE methods. However, for some downstream applications (notably immunoprecipitation) a gentler lysis method should be used.
This protocol refers to 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 cold sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 10% Glycerol, 100 mM DTT) at a 5×106 cells/ml sample buffer ratio.
3. Scrape the dish with a cell scraper and collect the lysate into a microtube.
4. Boil the sample at 100 ֯C for 5 minutes.
5. Sonicate the boiled sample for 5 seconds.
6. Centrifuge the sample at 14,000 rpm for 5 minutes.
7. Transfer the supernatant to a clean tube and store at -80°C until further use.
Note: Cell count can be established by dislodging the cells with trypsin from a parallel plate and counting them.