| Assay Method Information | |
| | IRAK4 Kinase Activity Assay |
| Description: | The compounds were prepared with DMSO to 100 times the final reaction concentration and diluted to 10 concentrations in sequence at a 3-fold dilution ratio starting from 1 μM. Then 0.25 μL was transferred to a 384-well plate using Echo550. A 1× kinase buffer (50 mM HEPES, pH=7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT) was used to prepare a kinase solution of 2.5 times the final concentration. Then 10 μL of the kinase solution of 2.5 times the final concentration was added to each compound well, shaken and mixed uniformly, and incubated at room temperature for 10 min. A 1× kinase buffer was used to prepare a mixed solution of ATP and substrate Kinase Substrate 8 with a concentration of 25/15 times the final concentration. 15 μL of the mixed solution of ATP and the substrate with a concentration of 25/15 times the final concentration were added to each well (the final concentration of IRAK4 kinase was 1 nM, the final concentration of the substrate was 3 μM, and the final concentration of ATP was 15.6 μM), shaken and mixed uniformly, and reacted at room temperature for 60 min. Finally, 30 μL of stopping solution (100 mM HEPES, pH=7.5, 0.0015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) was added to terminate the reaction. CaliperEZ Reader II was used to read data about conversion rates that was then converted into data about inhibition rates. |
| Affinity data for this assay | |
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