| Assay Method Information | |
| | BTK inhibition Assay |
| Description: | Reagent and Procedure by Reaction Biology Corporation: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. Required cofactors were added individually to each kinase reaction. Compound handling: testing compounds were dissolved in 100% DMSO to specific concentration. The serial dilution was conducted by Integra Viaflo Assist in DMSO.Reaction Procedure:The substrate was prepared in freshly prepared reaction buffer of above and required cofactors were added to the substrate solution. The kinase was delivered to the substrate solution and the mixture was gently mixed. Testing compounds in 100% DMSO was added to the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range) and the new mixture was incubated for 20 min at room temperature. Then 33P-ATP (Specific activity 10 μCi/μl) was added to the reaction mixture to initiate the reaction and the mixture was incubated for 2 hours at room temperature. The radioactivity was detected by filter-binding method and the kinase activity data was expressed as the percent of remaining kinase activity in test sample compared to vehicle (dimethyl sulfoxide) reaction. IC50 values and curve fits were obtained using Prism (GraphPad Software). |
| Affinity data for this assay | |
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