| Assay Method Information | |
| | BTK Enzyme Activity Assay |
| Description: | The specific experimental process of BTK enzyme activity test is as follows:Buffer: 20 mM hydroxyethylpiperazine ethylsulfuric acid (Hepes) (pH 7.5), 10 mM magnesium chloride, 1 mM ethylene glycol bisaminoethyl ether tetraacetic acid (EGTA), 0.02% polyoxyethylene lauryl 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. Configuring the substrate in the newly prepared reaction buffer2. Adding the required cofactors to the above substrate solution3. Adding the kinase BTKC481S to the above substrate solution and mixing well4. Adding the compound dissolved in DMSO to the kinase reaction mixture throughEcho550 (Acoustic technology; nanoliter range), and incubating at room temperature for 20 minutes5. Adding 33P-ATP (with a specific activity of 10 μCi/μL) into the reaction mixture to initiate the reaction6. Incubating at room temperature for 2 hours7. Detection of radioactivity by filtration-binding method8. Kinase activity data represent the percentage of remaining kinase activity in the test sample compared to the vehicle (dimethyl sulfoxide) reaction. Using Prism (GraphPad software) to obtain IC50 values and fitting curves |
| Affinity data for this assay | |
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