| Assay Method Information | |
| | Kinase Inhibitory Assay |
| Description: | The compounds were diluted in DMSO and configured into a solution with a final concentration of 10 mM; Dilute RIP1 kinase from 140 ng/μL to 20 ng/μL for use. Dilute 10 mM of ATP to 100 μM for later use; Dilute 5 mg/mL of MBP to 1 mg/mL for later use. Appropriate embodiments were added into the well of the 384-well plate, 10 μl 100M ATP was evenly mixed with 1 μl 1 mg/mL MB P, the mixed solution was added to the well, and 2 μL 20 ng/L RIP1 kinase was added to the hole, and then incubated at room temperature for 1 h. Subsequently, 5 μL ADP-Glo reagent was added to each well and incubated at room temperature for 40 min. Then, 10 μL ADP-Glo detection reagent was added to each well and incubated at room temperature for 30 min. Finally, the RLU value (relative light unit) of each well was measured by ATP fluorometer, and each sample was repeated twice. Inhibition rate=(RLU value of blank group−RLU value of drug administration group)/RLU value of blank group×100%. |
| Affinity data for this assay | |
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