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Assay Method Information |
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| | JAK Enzyme Assay |
| Description: | Test compounds were solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. Compounds were diluted in DMSO to create an 11-point half log dilution series with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 in the assay, resulting in a final assay compound concentration range of 10 μM to 100 pM and a final assay concentration of 1.7% DMSO. Test compounds and controls solubilized in 100% DMSO were added (250 nL) to a 384 well polypropylene plate (Matrical) using anon contact acoustic dispenser. Kinase assays were carried out at room temperature in a 15 μL reaction buffer containing 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20 and 1mM Dithiothreitol (DTT). Reaction mixtures contained 1 μM o |
| Affinity data for this assay | |
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