| Assay Method Information | |
| | Kinase Assay |
| Description: | The kinase activity of BTK (C481S) was assayed at Reaction Biology Corporation. The substrate in the BTK (C481S) reaction, pEY (poly[Glu:Tyr] (4:1)) (Sigma, Cat. #P7244-250MG), was prepared in fresh 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). BTK(C481S) (SignalChem, Cat. #B10-12CH) was delivered into the substrate solution and mixed gently. The final concentrations of BTK (C481S) and the substrate in the reaction mixture were 6 nM and 0.2 mg/ml, respectively. Compounds were tested in 10-point concentration/response mode with 3-fold serial dilution steps starting at 1 μM. Compounds in 100% DMSO were delivered into the kinase reaction mixture by acoustic liquid delivery technology (Echo550; nanoliter range) and incubated for 20 min at room temperature. 10 μM [33P]-ATP (ATP: Sigma, Cat #: A7699; [33P]-ATP: Hartmann Analytic, Cat #: SCF-301-12) was delivered into the reaction mixture to initiate the reaction. The mixture was incubated for 120 min at room temperature. Radioactivity was detected utilizing a proprietary filter-binding method. |
| Affinity data for this assay | |
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