protein kinase C alpha
PKCα (5-20 mU diluted in 20 mM Hepes pH 7.4, 0.03% Triton X-100) is assayed against Histone H1 in the presence of PtdSerine and DAG (0.1 mg/ml. and 10 µg/ml) and 0.1 mM CaCl2. The assay is carried out in a final volume of 25.5 µl containing 20 mM Hepes pH 7.4, 0.03% Triton X-100, 0.1 mg/ml Histone H1, 10 mM magnesium acetate and 0.02 mM[33P-γ-ATP] (50-1000 cpm/pmole) and incubated 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 mM orthophosphoric acid.PtdSer/DAG preparation:- PtdSer stock = 10 mg/ml in MeOH/Chloroform (1:2). Dry down required amount. Resuspend in appropriate volume of 10 mM Hepes pH 7.4. Vortex and briefly sonicate. (2 x 10-15 seconds at 10-15 seconds apart). DAG stock = 10 mg/ml in MeOH/chloroform (1:2). Dry down required amount. Add sonicated PtdSer solution. Vortex and sonicate.
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