| Assay Method Information | |
| | SIRT2 Enzyme Activity Assay |
| Description: | The SIRT2 enzyme activity assay is established based on the published studies. Firstly, peptide segment Ac-Gln-Pro-Lys-[Lys-(Ac)]-AMC, the substrate of SIRT2, was synthesized to serve as a substrate in the enzyme activity assay. The attached fluorescent label was AMC (7-Amino-4-methylcoumarin). The whole assay includes two steps: the catalytic reaction was carried out in 60 μL of reaction solution (25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 and 1 mg/mL BSA) with the addition of 500 μM NAD+, 50 μM of substrate peptide, 1.0 μg of SIRT2, and Compounds 1-18 with different concentrations. The reaction was carried out at 37° C. for 2 hours. In this reaction, the acetyl group of Lysine residue on the small molecule peptide segment was removed to varying degrees. Afterwards, 60 μL of sample treating solution (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, Trypsin and 4 mM nicotinamide) was added to the reaction solution and mixed, and placed at 37° C. for 20 min. Absorption intensity was measured by the enzyme-labeled instrument configured as an excitation light of 355 nm and an absorption light of 460 nm. |
| Affinity data for this assay | |
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