| Assay Method Information | |
| | BTK Kinase Assay |
| Description: | 1. Reaction conditions: buffer conditions: 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. Reaction procedure:2.1. Preparing an indicator substrate in a freshly prepared reaction buffer 2.2. Delivering the desired cofactor to the above substrate solution 2.3. Delivering the indicated kinase to the substrate solution and gently mixing same 2.4. Delivering the compounds in DMSO to the kinase reaction mixture using acoustic technology (Echo550) 2.5. Initiating the reaction (final concentration of ATP: 5 μM) by delivering 33P-ATP (final specific activity: 0.01 μci/μL) to the reaction mixture 2.6. Incubating the kinase reaction at room temperature for 120 minutes 2.7. Recording the reaction on P81 ion exchange paper (Whatman #3698-915) 2.8. Washing the filter widely with 0.75% phosphoric acid 2.9. Measuring the radioactive phosphorylated substrate remaining on the filter paper. 3.Data analysis: the kinase activity data is expressed as the percentage of the remaining kinase activity in a test sample compared to a reaction with a carrier (dimethyl sulfoxide), and IC50 values and curve fitting are obtained using Prism4 software (GraphPad). |
| Affinity data for this assay | |
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