| Assay Method Information | |
| | HTRF Displacement Assay |
| Description: | Test compounds were prepared as 10 mM stock solutions in DMSO (Fisher cat #BP231-100). Compounds to be assayed were dispensed using an Echo 650 acoustic dispenser (Beckman Coulter) on a 384 well plate in 6 doses applying four-fold dilutions from the highest concentration of 5 μM. N-terminal GST-tagged recombinant full-length BTK wildtype protein and BTK C481 S protein 2-659 were purchased from Carna Biosciences, Inc (Kobe, Japan). BTK wildtype or BTK C481S protein was separately incubated with compound in assay buffer containing kinase tracer 239 (Thermo Fisher) and Mab anti GST-Th (PerkinElmer). After a 1-hour incubation at room temperature, the HTRF signal was measured using SPARK plate reader (Tecan) using the TR fluorescence mode. The HTRF ratio is calculated for DMSO, no protein control and compound samples using the following equation: HTRF ratio=Emmision665/Emmision620×10{circumflex over ( )}4. The signal for the no protein control samples was used to subtract background noise from the DMSO and compound samples. Background subtracted HTRF ratios are used to calculate the percent inhibition, where DMSO controls are set to 0 percent inhibition. The percent inhibition is plotted as a function of compound concentration and IC50 values were calculated from a four-parameter logistic fit in Prism v9.4 (GraphPad), where the bottom and top are constrained to 0 and 100, respectively. |
| Affinity data for this assay | |
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