| Assay Method Information | |
| | Screening Assay Against IRAK4 with ATP |
| Description: | Table 2: Prepare Ix kinase base buffer and stop buffer for testing kinases: (1) base buffer: 50 mM HEPES (pH 7.5), 0.0015% Brij-35; (2) stop buffer: 100 mM HEPES (pH 7.5), 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA. Then prepare compounds for testing: Dilute the compound to 50× of the final desired highest inhibitor concentration in reaction by 100% DMSO. Transfer 100 μL of this compound dilution to a well in a 96-well plate (source plate).Serially dilute the compounds by 3-fold for a total of 10 points. Transfer 10 μL of the compound from the source plate to a new 96-well plate (intermediate plate) and add 90 μL of Ix kinase buffer. Shake the mixture on the intermediate plate for 10 min. Transfer 5 μL of each well from the 96-well intermediate plate to a 384-well plate in duplicates. Add 10 μL of 2.5× enzyme solution to each well of the 384-well assay plate and incubate at room temperature for 10 min. Add 10 μL, of 2.5×FAM-labeled peptide and ATP solution and incubate at 28° C. for a period of time. |
| Affinity data for this assay | |
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