| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | Kinase assays were performed at Reaction Biology Corp., Malvern, Pa., USA, using the following general procedure: 1) Prepare indicated substrate in freshly prepared Base Reaction Buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO). 2) Deliver cofactors (1.5 mM CaCl2, 16 ug/mL Calmodulin, 2 mM MnCl2) to the substrate solution above 3) Deliver indicated kinase into the substrate solution and gently mix 4) Deliver varying concentrations of test compound in DMSO into the kinase reaction mixture 5) Deliver 33P-ATP (specific activity 0.01 μCi/μL final) into the reaction mixture to initiate the reaction 6) Incubate kinase reaction for 120 min at room temperature 7) Reactions are spotted onto P81 ion exchange filter paper (Whatman #3698-915) 8) Unbound phosphate is removed by washing filters extensively in 0.75% Phosphoric acid. 9) 33P signal was determined using Typhoon phosphorimagers (GE Healthcare). After subtraction of background derived from control reactions containing inactive enzyme, IC50 values were determined using the nonlinear regression function in Prism (Graphpad software). |
| Affinity data for this assay | |
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