| Assay Method Information | |
| | ADP-Glo Kinase Assay |
| Description: | The kinase activity of recombinantly generated catalytic kinase (also known as JH1) domain of human JAK1, JAK2, JAK3 and TYK2 were evaluated in a plate-based assay using the ADP-Glo™ Kinase Assay platform. Specifically, 4 nM of recombinant JAK1 kinase domain is used to phosphorylate 50 μM of a JAK3-342 (sequence ALVDGYFRLTT) peptide in the presence of 35 μM ATP. Catalytic activities of recombinant JAK2, JAK3 and TYK2 kinase domain (0.2, 0.3 and 2 nM, respectively) are evaluated by the phosphorylation status of the JAK3-974 (50 μM; sequence LPLDKDYYVVR) peptide with the addition of ATP (15, 4 and 10 μM, respectively). The reactions proceed for 100 minutes and the catalytic activity is quantified by first depleting the unused ATP, converting the hydrolyzed ADP into ATP to generate luminescence in a luciferase reaction; which is the basis of the ADP-Glo platform. Compounds are tested at either 10 μM or 1 μM top concentration, 11 points of 3-fold dilution. |
| Affinity data for this assay | |
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