| Assay Method Information | |
| | BTK Kinase Assay |
| Description: | 1× buffer preparation (which was prepared and used immediately): Kinase assay buffer III was diluted with ddH2O to 1× assay buffer for later use.The compound to be tested was diluted with 100% DMSO to 10 μM as the first concentration, and followed by a 5-fold dilution to the 8th concentration using a multi-channel pipette, i.e., it was diluted from 10 μM to 0.128 nM.Each gradient of the compound to be tested was diluted into a working solution with 5% DMSO using 1× buffer, 1 μL/well was added to the corresponding well, and a double-replicate well experiment was set up. 2 μL of BTK enzyme (4 ng) was add to each well and incubating at 25° C. for 30 minutes. After the incubation was completed, 2 μL of a mixture of substrate and ATP (2 μM ATP, 0.2 μg/μL PolyE4Y1) was added to each well and incubating at 25° C. for 120 minutes. At this point, the final concentration gradient of the compound was diluted from 100 nM to 0.00128 nM. After the reaction was completed, 5 μL of ADP-Glo reagent was added to each well, and the reaction continued at 25° C. for 40 minutes. After the reaction was completed, 10 μL of kinase detection reagent was added to each well. After reacting at 25° C. for 30 minutes, PerkinElmer Nivo multi-label analyzer was used to read the chemiluminescence, and the integration time was 0.5 seconds. |
| Affinity data for this assay | |
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