The protein pellet was dissolved in caspase buffer (50 mM Hepes pH 7,2, 50 mM NaCl, 0,1% CHAPS, 10 mM EDTA, 5% glycerol and 10 mM DTT), 10 µl per 106 starting cells.
Cell lysis was performed with 50 mM Pipes-HCl, pH 6,5, 2 mM EDTA, 0,1% CHAPS, 10 mM NaF, 5 mM DTT and protease inhibitors.
Beads were washed twice with 0.5% CHAPS, 0.5% NP-40, and twice with 1% CHAPS, 1% NP-40.
All transfected cell pellets were re-suspended in CHAPS lysis buffer (TRAPEZE® 1× CHAPS Millipore Company).
The IP beads were washed 3 times with RIPA buffer and one time with 1× CHAPS buffer supplied by TRAPeze Telomerase Detection kit (Millipore, Billerica, MA, USA).
24 h post transfection, cells were lysed in 1× CHAPS buffer supplied by TRAPeze Telomerase Detection kit (Millipore, Billerica, MA, USA).
One million live cells were pelleted and lysed with 1× CHAPS buffer according to the TRAPeze kit (Upstate/CHEMICON) manufacturer instructions.
Cells were washed with 1xPBS and resuspended in ice-cold 1 % CHAPS lysis buffer (1 % CHAPS, 50 mM Tris [pH 7.6], 120 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, and 1 mM β-mercaptoethanol) with a cocktail of protease inhibitors (EMD Biosciences).
Serum samples were initially diluted 15-fold and tissue extracts to 1.5 mg protein per ml in 8 M urea +1% CHAPS.
For Western blot analysis, transfected or induced cells were maintained in the presence of 50uM z-VAD-fmk and harvested and lysed in 1% CHAPS buffer (150mM NaCl, 10mM HEPES [pH 7.4], 1% CHAPS) after the indicated time.
