| Assay Method Information | |
| | hACLY ADP-Glo Activity Assay |
| Description: | Table 3: Test compounds were 3-fold serially diluted in DMSO over 11-point concentration range and dispensed onto a 384-well plate. Recombinant human ACLY full length protein was purified. Concentrations of ACLY protein, sodium citrate, coenzyme A, and ATP in the reaction were optimized for standardized homogenous enzyme assay using ADP-Glo™ Kinase (Promega Inc.). The assay measured ADP formed from the enzymatic reaction.The reaction buffer consisted of the assay buffer (50 mM HEPES pH 8.0, 10 mM MgCl2, 4 mM 1,4-Dithiothreitol, 0.01% Brij® 35).ACLY protein (0.5 nM) was added to the prepared reaction buffer, and the mixture was dispensed into the assay plate and incubated for 30 minutes at room temperature. Next, 15 μM sodium citrate, 1 μM coenzyme A, and 80 μM ATP were added into the assay plate and incubated for 60 minutes at room temperature. The final reaction volume for each well was 5 μL.5 μL of ADP-Glo™ reagent was added, and the mixture was incubated for 40 minutes at room temperature. 10 μl ADP-Glo™ detection reagent was added, and the mixture was incubated for 30 minutes at room temperature. Luminescence was determined using EnVision microplate reader with ultra-luminescence module (Perkin Elmer Inc). Concentration-response curve-fitting and IC50 determination was performed using Xlfit Software (version 5.5.0) with a four-parameter logistic regression fit model. |
| Affinity data for this assay | |
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