| Assay Method Information | |
| | Determination of IRAK4 Kinase Activity |
| Description: | The following methods were used to determine the inhibition degree of the preferred compound of the present invention on IRAK4 kinase activity in vitro. In this evaluation, the HTRF KinEASE-STK S1 Serine/Threonine kinase kit produced by Cisbio was used to determine the phosphorylation degree of biotinylated polypeptide substrate by homogeneous time-resolved fluorescence technique (HTRF).Detailed methods can be referred to the kit instructions, and the experimental process was briefly described as follows. Firstly, the compounds of the present invention were dissolved in DMSO, and the final concentration was 10 mM. Then, the buffer solution provided in the kit was used for equal gradient dilution, so that the final concentration range of the tested compound in the reaction system was 16000 nM-0.008 nM, and the final concentration of DMSO is less than 2%.The adenosine triphosphate (ATP) concentration in the test was the corresponding ATP Km value (300 μM) determined in advance. Compounds, kinase, biotinylated polypeptide substrate and ATP were incubated at 37?? C. for 1 h for kinase reaction, then anti-phosphorylated Serine/Threonine antibody coupled with compound of europium element and modified XL665 streptavidin were added into the reaction system to terminate the reaction, and incubated at room temperature for 1 h. After incubation, the fluorescence intensity of each well at emission wavelengths of 615 nm and 665 nm was determined on the microplate reader FLUOstar Omega under the excitation wavelength of 337 nm in HTRF mode, and the Ratio value was calculated by using the formula Ratio=(665 nm/615 nm)??104. Compared with the fluorescence intensity ratio of the control group, the inhibition rates of the compound at each concentration were calculated, and then the IC 50 value of the compound was calculated by fitting the nonlinear curve of logarithmic concentration-inhibition rate with GraphPad Prism5. |
| Affinity data for this assay | |
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