| Assay Method Information | |
| | Biochemical Activity ACSS2 Assay |
| Description: | The biochemical activity assay for ACSS2 is based on the detection of released AMP by AMP-Glo Assay kit (Promega, Madison). The assay is run in three steps: the enzymatic reaction in which human rec ACSS2 activates acetate with ATP and Coenzyme A as cosubstrates to acetyI-coA thereby releasing AMP and the detection reaction of AMP by AMP Glo assay in which after the destruction of the residual ATP with AMP Glo reagent 1 produced AMP is converted to ATP that is measured in a luciferase assay system (detection reagent). The ACSS2 activity correlates with the detected luminescence signal. The assay was performed in Perkin Elmer 384well 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 1 h 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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