| Assay Method Information | |
| | TYRO3 Enzymatic Assay |
| Description: | Poly(Glu, Tyr) sodium salt (Glu:Tyr (4:1), quality level 200, molecular weight 5,000-20,000, obtained from Sigma Aldrich, catalogue #P7244, assay concentration 0.20 mg/mL) and recombinant human TYRO3 (obtained from ThermoFisher Scientific, catalogue #PV3828, assay concentration 2 nM) were mixed in assay buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO, with added MnCl2 at a final concentration of 2 mM). Compounds of interest (in DMSO, serial 3-fold dilution from 10 μM to 0.5 nM) or control (1% DMSO) were dispensed into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range). After incubation at room temperature for 20 minutes, the kinase reaction was initiated by addition of [32P]-ATP (Specific activity 10 μCi/μl) and the mixture was incubated at room temperature for 2 hours. The reaction was then stopped by spotting the reaction mixture on strips of phosphocellulose P81 paper. Following washing, the radioactivity of the P81 paper was measured and kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. |
| Affinity data for this assay | |
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