| Assay Method Information | |
| | MERTK, AXL, TYRO3 Kinase Homogeneous Time Resolved Fluorescence (HTRF) Assay |
| Description: | Binding assays contained 1 nM kinase (GST-MERTK 578-872, HIS-AXL 473-End, or GST-TYRO 451-890), 0.2 nM terbium labelled antibody (anti-GST or anti-HIS), fluorescein-labeled kinase tracer at its respective kinase Kd, and test compounds in assay buffer consisting of 20 mM Hepes pH 7.5, 10 mM MgCl2, 0.015% Brij-35, 2 mM DTT, and 50 μg/ml BSA. Assays were performed in black, flat-bottom, 1536-well plates. The reactions were incubated at room temperature for 60 minutes, following which the HTRF signals, ratio of fluorescence intensities at emission wavelengths for fluorescein acceptor (520 nm) and terbium donor (495 nm), the 520/495 ratio, were measured on the Envision Plate reader. Inhibition data were calculated from the 520/495 ratio generated by the no protein control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition, and dose response curves generated to determine the concentration required for inhibiting 50% of the HTRF signal (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. |
| Affinity data for this assay | |
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