| Assay Method Information | |
| | Kinase-Glo Plus Luminescence Kinase Assay |
| Description: | Method B conducts the kinase assay with the following condition and procedure: The assay was performed using Kinase-Glo Plus luminescence kinase assay kit (Promega). It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The compounds were diluted in 10% DMSO and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. All of the enzymatic reactions were conducted at 30° C. for 40 minutes. The 50 μl reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT, 0.2 mg/ml substrate peptide, 10 μM ATP and BTK. After the enzymatic reaction, 50 μl of Kinase-Glo Plus Luminescence kinase assay solution (Promega) was added to each reaction and incubate the plate for 5 minutes at room temperature. Luminescence signal was measured using a BioTek Synergy 2 microplate reader. Btk activity assays were performed in duplicate at each concentration. |
| Affinity data for this assay | |
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