| Assay Method Information | |
| | Experimental Method Screening Method of ACAT1 Small Molecule Inhibitors |
| Description: | ACAT1 can catalyze two molecules of acetyl coenzyme A to reversibly form acetoacetyl coenzyme A. Its activity was measured through the reaction between ACAT1-catalyzed substrate acetoacetyl-coenzyme A and coenzyme A, from which the product acetyl-coenzyme A is produced. Since acetoacetyl-coenzyme A is specifically absorbed in a specific spectrum, the impact on the enzyme activity of ACAT1 can be reflected through the detection of an increase or decrease of absorption in a specific spectrum.ACAT1 recombinant proteins were expressed and purified by Escherichia coli, and the concentration of ACAT1 recombinant proteins obtained was 1 mg/mL. The buffers adopted for ACAT1 small molecule inhibitor screening were 50 mM Tris-HCl (pH 8.1), 20 mM MgCl2, and 40 mM KCl. In 200 μL of enzyme catalytic system, 1 μL of ACAT1 recombinant proteins, the substrate acetyl-coenzyme A whose final concentration was changed from 25 μM to 100 μM through a CPM probe, and small molecule inhibitors at different concentrations were added, respectively. |
| Affinity data for this assay | |
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