| Assay Method Information | |
| | Biochemical Activity ACSS2 Assay |
| Description: | The assay was performed in Perkin Elmer 384 well white Proxiplates in a total volume of 8 μl. 1 nM (fc)C-term myc tagged ACSS2 (human, recombinant, Origene, Rockville, US) and a mixture of 100 μM (fc) ATP, 100 μM (fc) Coenzyme A and 500 μM (fc) sodium acetate were incubated in a total volume of 5 μl (50 mM Hepes, 1 mM Mg-chloride, 150 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, 0.3% DMSO, pH 7.5) in the absence or presence of the test compound (10 dilution concentrations, start conc 30 μM) for 180 min at 37 C. The reaction was stopped and residual ATP destroyed by the addition of 1 μl AMP Glo reagent solution (Promega, Madison, US). After 1h incubation at room temperature 2 μl of AMP Glo detection reagent was added and the assay was incubated for 0.75 hr at room temperature. The luminescence signal was measured with an Envision multimode reader (Perkin Elmer LAS Germany GmbH) at 700 nm in luminescence mode. The full value used was the inhibitor-free reaction. The pharmacological zero value was generated by addition of ACSS2 inhibitor (Ac-CoA Synthase Inhibitor CAS 508186-14-9-Calbiochem) in a final concentration of 5 μM. The inhibitory values (IC50) were determined using the program Assay Analyzer from GeneData. |
| Affinity data for this assay | |
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