| Assay Method Information | |
| | RIP2 Kinase Assay |
| Description: | GST-tagged recombinant human RIPK2(25 ng) is incubated with 5 µL of compounds (0.5% DMSO), 5 µL of MBP (0.5 µg/µl) and 5 µL of ATP (25 µM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 50 µM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30° C. for 60 minutes. After the incubation, 25 µL ADP-Glo reagent was added, and the reaction was incubated at 30° C. for 40 minutes to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 µL per well of detection reagent. Luminescence was detected after 30 minutes room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software or SigmaPlot 13.0. |
| Affinity data for this assay | |
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