| Assay Method Information | |
| | 1RAK4 Kinase Activity Assay |
| Description: | The KinEASE-STK SI serine/threonine kinase kit (Cisbio) was used to test the inhibitory effect of the compounds on the IRAK4 kinase activity, and the method is specifically as follows: A compound was dissolved in dimethyl sulfoxide, and the solution was diluted in the buffer of the kit with an equal gradient, such that the final concentration range of the test compound in the reaction systems was 10,000 nM-0.038 nM. Then 2.5 nM 1RAK4 kinase (Carna), 1 μM biotinylated polypeptide substrate (Cisbio) and 7 μM adenosine triphosphate (Sigma-Aldrich) were sequentially added, and the mixtures were incubated at 37° C. for 120 min. Subsequently, an anti-phosphoserine/threonine antibody (Cisbio) conjugated with a europium element compound and conjugated modified XL665 streptavidin (Cisbio) were added to the reaction systems to stop the reactions. After 1 h of incubation at room temperature, the fluorescence intensity of each well was measured at the excitation wavelength of 337 nm and the emission wavelengths of 620 nm and 665 nm with a microplate reader EnVision (PerkinElmer) in the HTRF mode, and Ratio values were calculated using the formula Ratio=(665 nm/620 nm)×10s. The inhibition rate of the compound at each concentration was calculated by comparing the fluorescence intensity ratio with that of the control group. |
| Affinity data for this assay | |
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