| Assay Method Information | |
| | High ATP Caliper Endpoint Assay |
| Description: | JAK3 enzyme (Invitrogen) stock solution is made up at 4.1 uM in sterile water. JAK3 enzyme stock is diluted to 2 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). ATP is made up at 10 mM stock in sterile water and diluted to 800 uM in assay buffer. Peptide (American peptide company) is made up at 30 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 1 nM. Compound and enzyme are pre-incubated for 60 minutes at room temperature using low evaporation lids before addition of 10 ul ATP/peptide mixture in assay buffer. |
| Affinity data for this assay | |
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