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Assay Method Information

Assay Name:  Kinase Assay
Description:   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) was diluted with 100% DMSO 500 times to a concentration of 20 μM, the dilute compound solution was diluted 1:3 in succession in a 384-well dilution plate (3657, corning) to gradient concentrations of 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 added to the 384-well reaction plate (prepared in step a), the plate was centrifuged at 1000 rpm and incubated at 25 C. for 15 minutes; d. 0.5 μL/well of substrate mixture (ATP (10 mM), Acetyl-CoA (2 mM), NaHCO3 (1000 mM)) was transfered into the 384-well reaction plate, the 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 is 1 nM. e. 10 μL/well of ADP-Glo solution was transfered into the 384-well reaction plate, the plate was centrifuged at 1000 rpm and incubated at 25 C. for 40 minutes. f. 20 L/well of kinase detection reagent was transfered into the 384-well reaction plate, the plate was centrifuged at 1000 rpm and incubated at 25 C. for 40 minutes. g. Relative Light Units (RLU) was read on an Envision multifunction plate reader. The signal intensity represents the level of ACC1/ACC2 kinase activity.
Affinity data for this assay
 

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Last update November 1, 2007
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