| Assay Method Information | |
| | High ATP Caliper Endpoint Assay |
| Description: | JAK1 enzyme (Invitrogen) stock solution is made up at 5.2 uM in sterile water. JAK1 enzyme stock is diluted to 20 nM in assay buffer (10 mM HEPES free acid pH 7.5, 10 mM HEPES free base pH 7.5, 10 mM MgCL2, 0.0005% Tween-20, 0.01% BSA) containing 2 mM DTT (all supplied by Sigma) with the addition of one protease tablet per 25 mls buffer (Roche). ATP is made up at 10 mM stock in sterile water and diluted to 5 mM in assay buffer. Peptide H236 (Caliper Life Sciences) is made up at 1.5 mM in 100% DMSO and diluted to 3 uM in assay buffer. Stop buffer comprises 140 mM HEPES, 22.5 mM EDTA (Sigma) and 0.15% coating reagent (Caliper Life Sciences).Assays are performed in Greiner polypropylene 384 well plates. Following compound preparation within the plate 10 ul of enzyme in assay buffer containing DTT is added using a Multidrop Micro. Final assay concentration of enzyme is 10 nM. Compound and enzyme are pre-incubated for 30 minutes at room temperature. |
| Affinity data for this assay | |
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