| Assay Method Information | |
| | BTK Kinase Activity Inhibition Test in Vitro |
| Description: | Kinase Reaction Process: (1) Preparation of 1 × Kinase buffer.(2) Preparation of compound with gradient concentrations: The test compound was tested at a concentration of 1 µM, diluted to a 100-fold final concentration of 100% DMSO solution in a 384 source plate, and 3-fold diluted to 10 concentrations. 250 nL of the compound with 100-fold final concentration was transferred to a destination plate OptiPlate-384F by using a liquid handler Echo 550.(3) A kinase solution with 2.5-fold final concentration was prepared with 1 × Kinase buffer.(4) 10 µL of the kinase solution with 2.5-fold final concentration was added to compound wells and positive control wells; and 10 µL of 1 ×Kinase buffer was added to negative control wells.(5) After centrifugation at 1000 rpm for 30 seconds, the reaction plate was shaken to mix well and incubated at room temperature for 10 min.(6) A 5/3-fold final concentration of a mixed solution of ATP and Kinase substrate 2 was prepared with 1 × Kinase buffer.(7) 15 µL of the mixed solution of ATP and substrate with 5/3-fold final concentration was added to start the reaction.(8) The 384-well plate was centrifuged at 1000 rpm for 30 seconds, shaken to mix well, and incubated at room temperature for 10 min.(9) 30 µL of a stop solution was added to stop the kinase reaction, and after centrifugation at 1000 rpm for 30 seconds, the plate was shaken to mix well;(10) The conversion rate was read with Caliper EZ Reader. |
| Affinity data for this assay | |
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