CHEMBL5739038

TYK2 Pseudokinase Domain Binding Assay: A binding buffer contains 20 mM Hepes pH 7.5, 150 mM NaCl, 10 mM MgCl, 0.015% Brij35, 2 mM DTT, 0.625 mM EGTA and 100 mM KF. The JH2 domain of TYK2 (amino acids 556-871 within the full-length protein) is expressed and purified by at Tsinghua University protein purification and identification platform. The test compound is dissolved to 0.1 mM in DMSO, followed by a serial 4-fold dilution with DMSO to a minimum concentration of 61 nM. Each concentration is further diluted 40-fold with the reaction buffer. To a 384-well assay plate (Corning, Cat. No. 4512) are added 5 μL of compound solution and 5 μL of TYK2 JH2 domain solution (160 nM). The mixture is incubated at room temperature for 30 minutes, and then added with 10 μL of a mixture of fluorescein-labeled probe (ThermoFisher, Cat. No. PV5593) (20 nM) and GST-Europium (Eu)-labeled antibody (Cisbio, Cat. No. 61GSTKLA) (40 ng/mL). After further incubation at room temperature for 30 minutes, the HTRF signal (ratio of fluorescence intensity at the emission wavelength of 615 nm and 665 nm for the fluorescein acceptor and the Europium donor, respectively) is measured on an Envision plate reader (Perkin Elmer).