| Assay Method Information | |
| | Inhibitory Activities of Compounds of the Present Disclosure Against RIPK1 Kinase (ADP-Glo Method) |
| Description: | The ADP GLO method was used for detecting the inhibitory activities of the compounds of the present disclosure against RIPK1 kinase. The method is specifically as follows:1. Reagents and Instruments1) RIPK1 kinase reaction system (containing RIPK1 kinase, MBP, 5× reaction buffer, MnCl2 and DTT) (Promega, VA7592)2) ADP-Glo kinase detection kit (containing ADP-Glo reagent and kinase detection reagents) (Promega, V9101)3) ATP solution (10 mM) (Thermofisher PV3227)4) 96-well small-volume white plate (Cisbio, 66PL96100)5) PHERA star microplate reader (BMG labtech)2. Experimental Method2.1 Reagent Preparationa. Reaction buffer: a reaction buffer containing DTT at a final concentration of 50 μM and MnCl2 at a final concentration of 4 mM was prepared using 5× reaction buffer;b. RIPK1 kinase solution: a RIPK1 kinase solution at a final concentration of 10 ng/μL was prepared using the reaction buffer;c. Mixed substrate of ATP and MBP: ATP at a final concentration of 60 μM and MBP at a final concentration of 0.6 μg/μL were separately prepared using the reaction buffer, and the prepared ATP and MBP were mixed in equal volumes;d. Compound: the compound was 3-fold diluted from an initial concentration of 33.3 μM to 9 gradient concentrations. All concentrations of compounds were 33.3-fold diluted with the reaction buffer for later use.2.2 Experimental Procedurea. The prepared RIPK1 enzyme solution was added to a 96-well plate at 2 μL/well, and 2 μL of the reaction buffer was added to column 1 instead of the enzyme.b. 2 μL of the compound was added to each well, DMSO was used as a control and added to column 1 and the last column instead of the compound, and the plate was centrifuged, shaken for 2 min, and incubated at room temperature for 10 min.c. 2 μL of the mixed substrate of ATP and MBP was added to each well, and the plate was centrifuged, shaken for 2 min, and incubated at room temperature for 90 min.d. 6 μL of the ADP-Glo reagent was added to each well, and the plate was centrifuged, shaken for 2 min, and incubated at room temperature for 40 min.e. 12 μL of the kinase detection reagent was added to each well, and the plate was centrifuged, shaken for 2 min, and incubated at room temperature for 40 min.f. The plate was read using the PHERA star microplate reader, and relative luminescence unit (RLU) values were recorded. g. Plotting was performed using the Graphpad software, and the IC50 values of the compounds of the present disclosure were calculated. |
| Affinity data for this assay | |
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