| Assay Method Information | |
| | Potency of IRAK4 Inhibitor Compounds in cKit Enzyme Assay |
| Description: | Potency of IRAK4 Inhibitor Compounds in cKit Enzyme Assay. Evaluation of the effects of the IRAK4 inhibitor compounds on the activity of the human cKit kinase quantified by measuring the phosphorylation of the substrate Ulight-TK peptide using a human recombinant enzyme and the LANCE detection method at Eurofins CEREP, catalog item 3070, SOP no 1C768: The test compound, reference compound or water (control) are mixed with the enzyme (0.38 ng) in a buffer containing 40 mM Hepes/Tris (pH 7.4), 0.8 mM EGTA/Tris, 8 mM MgCl2, 1.6 mM DTT and 0.008% Tween 20. Thereafter, the reaction is initiated by adding 100 nM of the substrate Ulight-TK peptide and 50 μM ATP, and the mixture is incubated for 30 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phopho-PT66 antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at lex=337 nm, lem=620 nm and lem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC50 value is calculated. |
| Affinity data for this assay | |
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