| Assay Method Information | |
| | Cellular Assays for Determining the Inhibition of Janus Kinases |
| Description: | To assay JAK1 inhibition, 2 μL of IL-6 diluted at 400 ng/mL (4×) in HBSS containing 0.1% BSA was added to the treated cells. The cells were then incubated for 30 min of cytokine treatment and lysed at the end by addition of 2 μL of 5×AlphaLISA SureFire Ultra lysis buffer from Perkin Elmer. The plate was agitated on a plate shaker for 10 min at room temperature. The cellular JAK1 activity was determined by measuring STAT3 phosphorylation at Tyr705 in lysates using the AlphaLISA SureFire Ultra kit (Perkin Elmer, Cat. No. ALSU-PST3-A500). A volume of 5 μL of acceptor mix and donor mix was added sequentially to lysates following the product protocol. The assay signals were recorded on EnVision plate reader (Perkin Elmer) with AlphaScreen optics setting. |
| Affinity data for this assay | |
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