| Assay Method Information | |
| | BTK Enzyme Activity Assay |
| Description: | The specific assay process of BTK enzyme activity assay is as follows: Buffer: 20 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (Hepes) (pH 7.5), 10 mM magnesium chloride, 1 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 0.02% polyoxyethylene dodecyl ether (Brij35), 0.02 mg/mL BSA, 0.1 mM sodium vanadate (Na3VO4), 2 mM dithiothreitol (DTT), 1% DMSO, 200 μM adenosine triphosphate (ATP).1. The substrate was prepared in freshly prepared reaction buffer;2. The required cofactor was added to the above-mentioned substrate solution;3. The kinase BTKC481S was added to the above-mentioned substrate solution and mixed well;4. The compound dissolved in DMSO was added to the kinase reaction mixture by Echo550 (Acoustic technology; nanoliter range), and the mixture was incubated at room temperature for 20 minutes;5. 33P-ATP (specific radioactivity of 10 μCi/μL) was added to the reaction mixture to initiate the reaction;6. The mixture was incubated at room temperature for 2 hours;7. The radioactivity was detected by filter-binding method;8. Kinase activity data represented the percentage of remaining kinase activity in the assay sample compared to the vehicle (dimethyl sulfoxide) reaction. IC50 values and fitted curve were obtained using Prism (GraphPad software). |
| Affinity data for this assay | |
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