CHEMBL3888822

Binding Assay: On the day at experiment Human Protein Kinase C betaII (PKCbII, Millipore cat#14-496) was incubated with 20 mM HEPES pH 7.4, 0.03% Triton X-100, 0.1 mM CaCl2, 0.1 mg/mL phosphatidylserine, 0.1 mg/mL histone H1, 10 mM MgAcetate. Amount of enzyme in each assay was 1.8 ng (final assay volume is 25 μL). The compounds were then diluted to 500 μM, and then serially diluted in 100% DMSO in semi-logarithmic decrements for 12 points. 0.5 μL of each concentration, in duplicate, was pipetted into a dry 96 well assay plate using a TIP Mosquito. Control and blank wells received 0.5 μL of 100% DMSO instead of compound. 14.5 μl kinase in assay buffer was added to each well. The assay was started with the addition of 10 μL ATP containing γ-33P-ATP] (specific activity approx. 500 cpm/pmol), to a final assay concentration of 70 μM. The reaction was allowed to proceed at room temperature for 40 minutes before the addition of 5 μL 3% ortho-phosphoric acid. Blank wells were acid blanks, and had 5 μL 3% ortho-phosphoric acid added before the addition of ATP. 10 μL of the stopped reaction products was transferred to a P30 filtermat which was then washed 4 times in 75 mM ortho-phosphoric acid, and once in methanol before drying. The filter was read by liquid scintillation counting using a Wallac Trilux.