CHEMBL5732154

IRAK4 Enzymatic DELFIA Assay, Protocol B: This is an in vitro assay to measure IRAK4 enzymatic activity utilizing the DELFIA (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay, Perkin-Elmer) platform, with inactive, unphosphorylated (0-phos), human IRAK4 FL (Full Length) construct to characterize IRAK4 inhibitor and control compounds at 600 μM ATP (KM). The final amount of enzyme in the assay is 0.1 nM inactive, 0-phos, IRAK4 FL, final concentration of substrate is 50 nM, and final concentration of DMSO is 2.5%.The test compound is solubilized in DMSO to a stock concentration of 30 mM. The dose response plates were prepared with a 4 mM primary compound concentration, serialized in DMSO and spotted (1 μL) into 384-well polypropylene plates as before.To begin the assay, 19 uL of reaction mixture containing 20 mM HEPES pH=7.5, 5 mM MgCl2, 0.0025% Brij-35, 600 uM ATP, 0.21 nM inactive, 0-phos, full-length recombinant human IRAK4 (GenBank ID AF445802) were aliquoted into the polypropylene plates containing 1 μL of test compound as before. 20 uL of 20 mM HEPES pH=7.5, 5 mM MgCl2, 0.0025% Brij-35, 600 μM ATP, and 100 nM ERM-biotinylated peptide (AGAGRDKYKTLRQIR) was added to start the reaction, which was run for 90 minutes at RT and stopped by the addition of 20 μL 0.3M EDTA.50 μL of the reaction mixture was transferred to a streptavidin-coated detection plate (DELFIA streptavidin coated plates, 384-well, white plates, Perkin-Elmer Life Sciences) and incubated for 30 minutes at RT. The plates were washed 4× with 75 μL per well of PBS containing 0.05% Tween-20. Plates were then incubated with 50 μL per well of antibody cocktail of Anti-pERM antibody at 0.125 μg/mL (Cell Signaling Technology), plus Anti-Rabbit IgG EuN1 at 0.25 μg/ml (Perkin-Elmer Life Sciences) in a solution of 10 mM MOPS pH=7.5, 150 mM NaCl, 0.05% Tween-20, 0.02% NaN3, 1% BSA, 0.1% Gelatin for 45 minutes. The plates were then washed as before. 50 μL per well of DELFIA Enhancement Solution (Perkin-Elmer Life Sciences) was added to the plate and incubated for 15 minutes at RT prior to being read on an Envision Model 2104 multi-label reader using a 340 nm excitation wavelength and a 665 nm emission wavelength for detection.