| Assay Method Information | |
| | Kinase Inhibitory Activity Assay (ATP 1mM) |
| Description: | Test method: Firstly, test compounds were prepared into 10 mM stock solution with DMSO, and continuously diluted into 10 concentrations at a 3-fold gradient for later use. 5X reaction buffer was diluted with deionized water into 1×reaction buffer (50 mM HEPES pH 7.5, 0.01% Brij-35, 10 mM MgCl2, 1 mM EGTA), which was used to prepare a mixture of kinase and peptide substrate, as well as a phosphorylated peptide substrate solution. The kinase concentration was determined based on enzyme titration, and the final concentration of peptide substrate and phosphorylated peptide substrate was 2 μM. 5 μL of mixture substrate of kinase and peptide substrate was added to each well on the 384 well plate. Then 5 nL of the test compound (starting with a final concentration of 10 μM) was added with an Echo520 Sampler. After shaking and mixing at room temperature for 15 minutes, added an appropriate amount of ATP to the well to meet the test requirements (note: according to the concentration recommended in manufacturer's instructions, the ATP concentration for JAK1 kinase test is 75 μM, 25 μM for JAK2 kinase test, M for JAK3 kinase test, and 25 μM for TYK2 kinase test). An additional 100% phosphorylation well (5 μL phosphorylated peptide substrate solution), a 0% phosphorylated well (5 μL of a mixture of kinase and peptide substrate, without test compounds and ATP), and a 0% inhibitory well (5 μL of a mixture of kinase and peptide substrate and appropriate concentration of 1mM ATP) were set as control. Two repeats were set for each concentration. |
| Affinity data for this assay | |
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