protein kinase C gamma
PKCgamma (5-20 mU diluted in 20 mM Hepes pH 7.4, 0.03% Triton X-100) is assayed against Histone H1in the presence of PtdSerine and DAG (0.1 mg/ml. and 10 μg/ml) and 0.1 mM CaCl2. The assay iscarried out in a final volume of 25.5 μl containing 20 mM Hepes pH 7.4, 0.03% Triton X-100, 0.1mg/ml Histone H1, 10 mM magnesium acetate and 0.02 mM[33P-γ-ATP] (50-1000 cpm/pmole) andincubated for 30 min at room temperature. Assays are stopped by addition of 5 μl of 0.5 M (3%)orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mMorthophosphoric acid.PtdSer/DAG preparation:- PtdSer stock = 10 mg/ml in MeOH/Chloroform (1:2). Dry down requiredamount. Resuspend in appropriate volume of 10 mM Hepes pH 7.4. Vortex and briefly sonicate. (2 x10-15 seconds at 10-15 seconds apart). DAG stock = 10 mg/ml in MeOH/chloroform (1:2). Dry downrequired amount. Add sonicated PtdSer solution. Vortex and sonicate.
PKC_gamma_screening.pdf (91.81 KB)