| Assay Method Information | |
| | RIP1 kinase assay |
| Description: | RIP1 kinase assays were performed using ADP-Glo assay (Promega) according to manufacturer's protocol. Reactions were performed in 50 mM HEPES, pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.05% bovine serum albumin (BSA), 0.02% CHAPS buffer, containing 20 ng recombinant GST-human RIP1 kinase domain (amino acids 1-327), 50 mM ATP and 10 serial dilutions of inhibitors. Recombinant GST-RIP1 was generated using baculoviral expression system in Sf9 cells. Protein was purified by glutathione affinity chromatography, followed by size exclusion chromatography. Reactions were performed for 4 hr at room temperature and stopped by incubation with ADP-Glo reagent for 40 min at room temperature. Luminescent signal was developed by incubation with Kinase Detection reagent for 30 min at room temperature. Signal was determined using Victor3V platereader (Perkin Elmer). Non-linear regression to calculate EC50 values was performed using GraphPad Prism software package. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |