| Assay Method Information | |
| | Biochemical Assay |
| Description: | The 2-hour 1 mM ATP Biochemical Assay employed a MesoScale Detection (MSD) format. The kinase reaction was based on the IRAK4 phosphorylation of a biotin labeled peptide (IRAK1 activation loop sequence 360-389).The kinase reaction in 30 μl was carried out in wells of a 384 well polypropylene assay plate, with 0.1 nM IRAK4, 1.6 μM of biotinylated peptide substrate and 1 mM ATP in 50 mM Hepes, pH 7.5, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% BSA, and 1% DMSO (from compound DMSO stocks), for 2 hour at room temperature. The activity was quenched with 11 μl of 70 mM EDTA, pH 8.To detect the phosphorylated biotinylated peptide substrate, 30 μl of the quenched reaction mixture was added to equivalent wells of a 384 well streptavidin coated MesoScale plate (Meso Scale Discovery #L21SA-1). After a 1 hour incubation of the plate for 1 hour at room temperature with gentle mixing, the plate wells were washed 3 times with 50 mM Tris, pH 7.5, 150 mM NaCl, 0.02% Tween-20.A 25 μl volume of 1:500 anti-P-Threonine Rabbit polyclonal Antibody plus 1:500 Goat-anti-Rabbit Sulfo Tag Antibody (Meso Scale Discovery R32AB-1) in 50 mM Tris, pH 7.5, 150 mM NaCl, 0.02% Tween-20 plus 2% BSA was then added to each well. After a 1-hour incubation of the plate for 1 hour at room temperature with gentle mixing, the plate wells were washed, 3 times with 50 mM Tris, pH 7.5, 150 mM NaCl, 0.02% Tween-20. A 40 μl volume of 2×MSD Read Buffer (Meso Scale Discovery R92TC-1) was added to each well, and the plate was read immediately in an MSD Plate Reader (Meso Scale Discovery). |
| Affinity data for this assay | |
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