| Assay Method Information | |
| | RIPK1 Kinase Activity Assay |
| Description: | ADP-Glo Kinase kit can be used to measure ADP level in kinase activity assay so as to distinguish the inhibitory effects of different compounds on RIPK1 kinase activity. The ADP-Glo assay was usually divided into three steps. Firstly, the kinase converted ATP to ADP and phosphorylated the substrate at the same time; secondly, ATP digestion reagent was added to degrade all ATP in the reaction system; finally, a detection reagent was added to reduce ADP to ATP, and energy from ATP was transferred to fluorescein which thus emitting a chemical luminescence that can be detected. Assay procedure can refer to manufacture's technical manual.(2) Reagent Preparation:1.33× kinase buffer: 5× kinase buffer stock (250 mM of NaCl, 150 mM of MgCl2, 2.5 mg/ml BSA (bovine serum albumin), 0.1% CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate), and 5 mM of dithiothreitol) was diluted with water to 1.33× kinase buffer;RIPK1 enzyme solution: the kinase was dissolved in 1.33× kinase buffer to make 40 nM as the final working concentration;ATP solution: 10 mM ATP stock solution in water was dissolved in 1.33× kinase buffer to make 10 μM as the final working concentration;4× compound preparation: the compound was diluted in a 3-fold gradient, and finally 4% DMSO aqueous solution containing different concentrations of the compound was obtained. The final concentrations of the test compound were 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.014, and 0.005 μM.(3) Specific Steps of the Experiment:The assay set up two control groups, one was 100% inhibition group (no enzyme treatment), another was 0% inhibition group (no inhibitor treatment), each control group contained 8 replicate wells. 2.5 μl of serially diluted compound was added to each well of a 384-well plate, with double replicate wells, and 4% DMSO solution was added to control wells. Then 5 μl of RIPK1 enzyme solution was added to each well except 100% inhibition control, 5 ul of buffer was added to 100% inhibition control, after that, 2.5 μl of ATP solution was added to all wells, the plate was vibrated at 1000 rpm for 30 seconds to perform transient centrifuge; finally the 384-well assay plate was put in the shaking incubator, and incubated at room temperature for 3 hours. After the enzymatic reaction was completed, 5 μl of ATP depletion reagent was added to each well, the mixture was centrifuged transiently, then the 384-well assay plate was put in the shaking incubator, and incubated at room temperature for 1 hour. 5 μl of ADP detection reagent was added to each well, and the mixture was centrifuged transiently, and incubated at room temperature for 0.5 hour. |
| Affinity data for this assay | |
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