CHEMBL3706262

In Vitro Kinase Inhibition Assays: All kinase assays were performed in 96-well FlashPlates from Perkin Elmer/NEN (Boston, Mass., USA) in a 50 μl reaction volume. The reaction mixture was pipetted in four steps in the following order:20 μl of assay buffer (standard buffer),5 μl of ATP solution (in H2O),5 μl of test compound (in 10% DMSO),10 μl of substrate/10 μl of enzyme solution (premixed),The assay for all enzymes contained 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-Orthovanadate, 1.2 mM DTT, 50 μg/ml PEG20000, 1 μM [-33P]-ATP (approx. 5×1005 cpm per well).