| Assay Method Information | |
| | Biological Assays against IRAK4 |
| Description: | To measure the IC50 values of compounds herein against IRAK4, a Z′-LYTE assay (ThermoFisher) was used. Briefly, 2.5 μL. of different concentrations of the compounds in 1% DMSO were added to 2.4 μL kinase buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA) in each well of a 384-well plate (Corning Cat. #3676). 5 μL of 2×IRAK4/Ser/Thr 07 mixture (prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MnCl2, 2 mM OTT, and 0.02% NaN3) and 2.5 μL of 4×ATP solution (4×ATP, 50 mM HEPES, pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA) were added to each well. The plate was shaken for 30 seconds, and then incubated at room temperature for 60 minutes. 5 μL of a 1:100000 dilution of Development Reagent A was added to each well. The plate was shaken for 30 seconds and incubated for 60 minutes at room temperature. The plate was subsequently read on a fluorescence plate reader, and the emissions ratio was calculated to determine the ratio of Ser/Thr 07 phosphorylated by the reaction. Emissions Ratio=Coumarin Emission (443 nm)/Flourescein Emission (520 nm). |
| Affinity data for this assay | |
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