| Assay Method Information | |
| | BTK and BTK C481S Enzyme Assay |
| Description: | A 3-fold gradient concentration stock solution of 1000× compound was prepared using DMSO and diluted 100-fold to 10× compound stock solution using reaction buffer (50 mM HEPES, pH 7.5, 0.0015% Briji-35, 2 mM DTT, 10 mM MgCl2), and the 10× compound stock solution was transferred to a 384-well plate. Enzyme reactions were set up with BTK Kinase Enzyme System (Promega Catalog #V2941) or BTK (C481S) Kinase Enzyme System (Promega Catalog #VA7033). First, a 2× enzyme solution containing 10 nM BTK or 10 nM BTK C481S was prepared with reaction buffer and added to the plate, and incubated with the compound for 10 minutes. Then, a 2.5× substrate solution containing ATP (125 μM) and Poly(Glu4, Tyr1) (0.05 μg/μL) was prepared with reaction buffer and added to the plate, and reacted at 20° C. for 90 minutes. Finally, the kinase activity was detected according to the experimental steps provided by ADP-Glo kinase Assay Kit (Promega, #V9101), and finally the luminescence chemiluminescence value was read. DMSO was used as the maximum signal value, and adding no enzyme was used as the minimum signal value. |
| Affinity data for this assay | |
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