| Assay Method Information | |
| | IRAK4 ADP-Glo Assay |
| Description: | The ADP-Glo reagents were thawed at ambient temperature. The Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate, and set aside.A stock volume of 5 Reaction Kinase Buffer was made with a final concentration of 100 mM MgCl2, 200 mM Tris-HCl, and 0.5 mg/ml of BSA, in distilled H2O with a final pH7.4. A 2 working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 μM DTT.The components of IRAK4 Enzyme were thawed on ice. Diluted IRAK4 in 1 Kinase Reaction Buffer (diluted from 2 buffer) was prepared at 5.0 ng/μl. A 250 μM working stock ATP Assay Solution was prepared in 1 Kinase Reaction Buffer (diluted from 2 buffer).The compound was diluted in DMSO from 250 μM in 4-fold series dilutions for 8 points. Then diluted 1:5 in 2 Reaction Buffer in a 96 well plate. 1.0 μl was transferred to a 384 well plate in duplicate. 2 μl of diluted Active IRAK4 was added (do not add to column 1) and 2 reaction buffer was added to column 1. 1 μl of 1 mg/ml stock solution of MBP substrate was added NOTE: MBP can be combined with ATP mix with equal volume and then added at 2 μl/well. Final reaction volume was 5 μl.The plate was centrifuged and the reaction mixture was incubated at room temperature for 60 minutes or at 30 C. for 30 minutes.The reaction was terminated and the remaining ATP was depleted by adding 5 μl of ADP-Glo Reagent. The 384-well plate was centrifuged and then the reaction mixture was incubated for another 40 minutes at ambient temperature. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |