CHEMBL3888821

Binding Assay: On the day at experiment Human Protein Kinase C delta (PKCδ, Millipore cat#14-504) was diluted in 20 mM HEPES, 0.03% Triton X-100. Amount of enzyme in each assay was 7.25 ng (final assay volume is 25 μL). All compounds for testing were diluted to 1 mM in 100% DMSO as an intermediary dilution. The compounds were then diluted further to 50 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 TTP Mosquito. Control and blank wells received 0.5 μL of 100% DMSO instead of compound. The assay was built with the addition of 14.5 μL of assay mixture, containing appropriately diluted enzyme and 20 mM HEPES pH 7.4, 0.03% Triton X-100, 0.05 mg/mL phosphatidylserine, and 50 μM ERMRPRKRQGSVRRRV. The assay was started with the addition of 10 μL ATP containing γ-33P-ATP] (specific activity approx. 500 cpm/pmol), to a final ass