| Assay Method Information | |
| | Biochemical kinase assay BTK C481 S |
| Description: | To determine the inhibitory activity of compounds on BTK C481S enzyme activity, the IMAP® assay (Molecular Devices) was used. Compounds were serially diluted in dimethylsulfoxide (DMSO) and subsequently in 4% DMSO in IMAP reaction buffer, which consists of 10 mM Tris-HCl, pH 15 7.5, 10 mM MgCl2, 0.01% Tween-20, 0.1% NaN3 and 1 mM freshly prepared dithiotreitol (DTT). Compound solution was mixed with an equal volume of full-length BTK C481S enzyme (Carna Biosciences, cat. no. 08-547) in IMAP reaction buffer. After pre-incubation of 1 hour in the dark at room temperature, fluorescein-labeled MBP-derived substrate peptide (Molecular Devices, cat. no. RP 7123) was added, followed by ATP to start the reaction. Final enzyme concentration was 1.2 nM final substrate concentration 50 nM, and final ATP concentration was 7 μM. The reaction was allowed to proceed for 2 hours at room temperature in the dark. The reaction was stopped by quenching with IMAP progressive binding solution according to the protocol of the manufacturer (Molecular Devices). Fluorescein polarization was measured on an Envision multimode reader (Perkin Elmer, Waltham, MA, U.S.A.). IC50 were calculated using XLfit™5 software (ID Business Solutions, Ltd., Surrey, U.K.). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |