CHEMBL3707732

In Vitro Kinase Activity Assay: Compounds to be tested were dissolved in 100% DMSO in a range of concentrations from 10-8 to 10-3 M including negative control (DMSO), then diluted with Kinase Buffer (50 mM HEPES, pH 7.5, 1 mM EGTA, 10 mM MgCl2, 0.01% Tween-20, and 2 mM DTT, added fresh) to 4x the final concentration. The final curve included 11 concentrations (10-11 to 10-5 M) plus negative control DMSO tested in triplicate.Kinase and ATP concentrations necessary for optimal activity were determined in separate experiments. (Table 2). Briefly, increasing concentrations of kinase were incubated in a fixed concentration of ATP (200 uM) in Kinase Buffer to determine maximal activity at various time points (30-120 min). The concentration of kinase that resulted in maximal activity with the shortest incubation time was then incubated in Kinase Buffer with increasing concentrations of ATP.