| Assay Method Information | |
| | JAK1, JAK2, and JAK3 Activity |
| Description: | Compounds described herein were tested for the ability to inhibit activity of human JAK1, JAK2 and JAK3, which was achieved using TR-FRET assays. Briefly, the Kinases JAK1 (2.5 nM), JAK2 (0.025 nM) and JAK3 (0.0125 nM) were incubated with a series of concentrations of the test compound in the presence of 1 mM JAK Common Substrate (biotin-ahx- EQEDEPEGDYFEWLE-CONH2), 2 nM Eu-labeled anti-pTYRPY20 and 80 nM Streptavidin APC. After 30 min incubation at RT, ATP (30, 5 and 5 mM respectively for JAK1, JAK2 and JAK3) was added to start the reaction, and incubated for 80 min at RT. The reaction was stopped by adding detection buffer and incubated for a further 60 min at RT. The samples were analyzed using Envision to calculate % inhibition at each of the series of concentrations of the test compound. The IC50 value of the compounds for each of the kinases were calculated using XLFit software. |
| Affinity data for this assay | |
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