| Assay Method Information | |
| | BTK-WT and BTK-C481S LanthaScreen |
| Description: | Inhibitory activity on BTK wild type (BTK-WT) and BTK Cys481Ser mutant (BTK-C481S) was measured using the LanthaScreen assay technology from ThermoFisher according to manufacturer's protocol.BTK-WT or BTK-C481 S (Genscript) were mixed and diluted with Eu-anti-GST antibody (Invitrogen) in Kinase buffer (50 mM Hepes pH 7.5+10 mM MgCl2+1 mM EGTA+0.01% Brij-35) to 15 and 6 nM, respectively. Final concentration in the assay for enzyme and antibody are 5 and 2 nM, respectively.Tracer (Kinase Tracer 236, Invitrogen) is diluted in Kinase buffer to 90 nM. Final concentration in the assay is 30 nM.Serial dilutions log 10 from 1 mM to 3.16 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 33-fold in Kinase buffer (50 mM Hepes pH 7.5+10 mM MgCl2+1 mM EGTA+0.01% Brij-35).The assay is performed as follows: 5 μL/well of BTK-WT or BTK-C481S enzyme and EU-anti-GST antibody dilution is mixed with 5 μL/well tracer dilution and 5 μL/well of compound dilution in Kinase buffer. Final compound concentration in the assay ranged from 10 μM to 0.316 nM, with 1% DMSO final concentration in assay. Mixture was incubated at room temperature in the dark and at different times of incubation (5 min, 10 min, 20 min, 30 min, 40 min, 60 min, 90 min, 120 min, 180 min and 300 min) the TR-FRET signal was read at 615 nm and 665 nm. The ratio 665/615 was used to calculate values expressed as percentage of the difference in readout (S/N) of the controls with and without Tracer. IC50 values for each timepoint were determined by curve fitting of the experimental results in Dotmatics. |
| Affinity data for this assay | |
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