| Assay Method Information | |
| | TYK2 Activity Inhibition Assay |
| Description: | The enzymatic activity of TYK2 was assessed by detecting the substrate phosphorylation level in a kinase reaction with a homogeneous time-resolved fluorescence (HTRF) kinase detection kit (Cisbio, 62TK0PEC). A reaction buffer contained an enzyme buffer (1×), 5 mM MgCl2, 1 mM DTT and 0.01% Brij35 from the kit; a human recombinant TYK2 protein (Carna Biosciences, 08-147) was diluted to a kinase reaction solution of 0.25 ng/μL with the reaction buffer; a substrate reaction solution contained 11.25 μM ATP and a biotin-labeled tyrosine kinase substrate diluted to 0.5 μM with the reaction buffer; a detection buffer contained 0.1 ng/μL Eu3+ labeled cage antibody (Cisbio, 61T66KLB) and 25 nM streptavidin-labeled XL665 (Cisbio, 610SAXLB) in the reaction buffer; the compound was dissolved to 10 μM in DMSO, followed by a serial 4-fold dilution with DMSO to a minimum concentration of 0.061 nM. Each concentration was further diluted 40-fold with the reaction buffer. To a 384-well assay plate (Corning, 3674) were added 4 μL of the compound solutions having a series of concentrations and 2 μL of kinase reaction solutions. After being mixed evenly, the mixtures were incubated at room temperature for 15 minutes, and then 4 μL of the substrate reaction solutions were added. |
| Affinity data for this assay | |
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