| Assay Method Information | |
| | Measurement of Human IRAK-4 Inhibitory Activity |
| Description: | For the measurement of the activity of the human IRAK-4 (Invitrogen, Cat. PV3362), phosphorylation of the IRAK-4 peptide substrate (biotin-KKKKRFSFKKSFKC) by the enzyme in the presence of 10 µM ATP (Sigma-Aldrich, Cat. A7699) was measured by the TR-FRET method. The enzymatic reaction was performed in a reaction buffer containing 50 mM HEPES (pH 7.2), 1 mM DTT, 0.1 mM Na3VO4, 5 mM MgCl2, 1 mM MnCl2, and 0.1% bovine serum albumin. For the measurement of the IRAK-4 inhibitory activity, a test compound was added to the reaction buffer containing 1 nM IRAK-4, 0.5 µM peptide substrate, and 10 µM ATP, and the mixture was incubated at 23° C. for 30 minutes. Then, a detection solution containing an antibody labeled with europium cryptate (0.3 µg/mL, the antibody was prepared by using the IRAK-4 peptide substrate as the antigen), streptavidin-XL665 (2 µg/mL, CisBio, Cat. 610SAXLB), 50 mM HEPES (pH 7.2), 0.1% BSA, 120 mM KF, and 66.7 mM EDTA (all the concentrations of the reagents are final concentrations) was added to terminate the reaction, and then the mixture was further incubated at 23° C. for 60 minutes. Fluorescence intensity was measured at wavelengths of 665 nm and 620 nm with a microplate reader, and the enzymatic activity was calculated as the ratio of fluorescence intensities at 665 nm and 620 nm (665 nm/620 nm). The IRAK-4 suppression ratio observed with addition of 12.5 µM staurosporine (LC Laboratories, Cat. S-9300) was defined to be 100%, the IRAK-4 suppression ratio observed with no addition of test compound was defined to be 0%, and IC50 of the test compound was calculated by using the 4-parameter logistic model of the data analysis software XLfit (ID Business Solutions Ltd.). |
| Affinity data for this assay | |
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