Irak4 kinase enzyme system

IRAK4 activity was measured using IRAK4 Kinase Enzyme System and ADP-Glo Kinase Assay (Promega, Madison, WI) with modifications. IRAK4 kinase was immunoprecipitated using Dynabeads Protein G as described above. After immunoprecipitation, IRAK4 kinase was eluted with 20 μl Elution buffer (50 mM Glycine pH2.8). For the functional assay, the pH of the eluate was adjusted by adding 20 μl of 1 M Tris (pH7.5). The final pH of the eluate was 7.5. In 96-well plate, the 25 μl total reaction volume included 5 μl 5X kinase buffer, 10 μl enzyme, and 10 μl substrate/ATP mix (2.5 μg MBP protein, 50 μM ATP, and 50 μM DTT). The kinase reaction was incubated at room temperature for 60 min. Then 25 μl ADP-Glo Reagent was added. Followed by incubation at room temperature for 40 min, 50 μl Kinase Detection Reagent was added. After incubation at room temperature for 30 min, the luminescence was recorded with 0.5 s Integration time using PerkinElmer Multilabel Counter.

IRAK4 Kinase activity was evaluated by the IRAK4 Kinase Enzyme System (V9421, Promega) according to the manufactorer’s instructions. Per manufactorer’s instructions, Staurosporine (S6942, Sigma) was administered to generate a kinase inhibitor dose-response curve.