| Assay Method Information | |
| | In Vitro Inhibitory Activity Test of Acetyl-CoA Carboxylase |
| Description: | The specific steps are as follows:a. 4.5 μL/well of ACC1/ACC2 working solution (2.22 nM) was added to a 384-well reaction plate (PerkinElmer, 6007290);b. The compound (10 mM stock solution) was diluted 500 times to 20 μM with 100% DMSO, and diluted in a ratio of 1:3 in a 384 dilution plate (3657, corning). The gradient concentrations of the compound were 20, 6.67, 2.22 0.74, 0.25, 0.082, 0.027, 0.009, 0.003, 0.001, 0 μM;c. 0.5 μL/well of the compound solution (prepared in step b) was transferred to the 384-well reaction plate (prepared in step a), then plate was centrifuged at 1000 rpm and incubated at 25° C. for 15 minutes;d. 5 μL/well of substrate mixture solution [ATP (10 mM), Acetyl-CoA (2 mM), NaHCO3 (1000 mM)] was transferred to the 384-well reaction plate, then plate was centrifuged at 1000 rpm and incubated at 25° C. for 30 minutes. The compound final gradient concentrations in the reaction system were 1000, 333.3, 111.1, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15, 0.05, 0 nM. The final concentration of DMSO was 5%; the final concentration of ACC1/ACC2 was 1 nM;e. 10 μL/well of ADP-Glo solution was transferred to the 384-well reaction plate, then the plate was centrifuged at 1000 rpm and incubated at 25° C. for 40 minutes;f. 20 μL/well of kinase detection reagent was transferred to the 384-well reaction plate, then the plate was centrifuged at 1000 rpm and incubated at 25° C. for 40 minutes;g. Relative luminescence unit (RLU) was read on an Envision multifunction plate reader. The signal intensity was used to characterize the activity of ACC1/ACC2 kinase. |
| Affinity data for this assay | |
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