| Assay Method Information | |
| | BTK Kinase Activity inhibition Assay |
| Description: | Activity of BTK protein kinase was tested by Caliper mobility shift assay (referring to J Biomol Screen 14:31, 2009). The compound of the present disclosure was dissolved in DMSO and then diluted 10-fold with kinase buffer solution (50 mM HEPES-pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT). 5 μl of 10% DMSO with 5-fold final reaction concentration of the compound dissolved was added into a 384-well plate, and a control well without the compound and a control well without kinase activity were respectively added 5 μl of 10% DMSO. 10 μl of BTK kinase solution (BTK, Cat. No. 08-080, Carna) with 2.5-fold final reaction concentration was added and mixed with the compound and then incubated for 10 minutes at room temperature, wherein the control well without kinase activity was added 10 μl of kinase buffer solution. 10 μl of a substrate solution of a substrate FAM-labeled SRCtide peptide (Biochem, Cat. No. 112394) and ATP (90 μM) with 2.5-fold final reaction concentration was further added to start the reaction. Then incubation was performed for 10 minutes at room temperature. After incubation for 1 hour 28° C., 25 μl of stopping solution (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) was added to terminate the reaction. The conversion ratio data was read on Caliper EZ Reader II (Caliper Life Sciences). The inhibition ratio was calculated and the calculation formula is: inhibition ratio %=(max-conversion)/(max-min)×100 %. |
| Affinity data for this assay | |
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