| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | The compound of the present invention and the control compound were dissolved in the assay buffer (50 mM Hepes pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.02% CHAPS, 0.5 mg/mL BSA), and diluted in a ratio of 1:1.5 using a 22-point titration (high concentration 2 μM), and then added to a 384-well plate. Each concentration of inhibitor (3.5 μL) and 3.5 μL of human RIP kinase (25 nM) dissolved in the assay buffer were added to the wells. After warming at 37° C. for 1 h, 3.5 μL of ATP (15 μM to 1.5 mM) in buffer was added to initiate the reaction, and then allowed to react at room temperature for 5 h. After completion of the reaction, 5 μL of ADP-Glo reagent containing 0.02% CHAPS was added to each well and incubated at room temperature for 1 h to terminate the kinase reaction and use up all remaining ATP. Then, 5 μL of ADP-Glo detection solution containing 0.02% CHAPS was added to each well and incubated at room temperature for 30 min to measure the emittance intensity. |
| Affinity data for this assay | |
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