| Assay Method Information | |
| | In Vitro Biological Assay |
| Description: | 50 uM of substrate peptide (acetylated AMC-labeled peptide from p53 residues 379-382, RHKKAc, BioMol. Cat. #KI-177), 91 nM of human SIRT1 (full length human Sirtuin 1 expressed in E. coli, BioMol. Cat. #SE-239) and 500 uM NAD+ in the assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 supplemented with 1 mg/ml BSA for dilution, BioMol. Cat. #KI-143) and 1% final concentration of DMSO were incubated in the presence of gradient concentrations of test compounds (10-dose with 3-fold serial dilution) at 30 C. for 2 h. The reactions were carried out in a 96-well microplate for fluorometry in a 50 ul reaction volume. After the deacetylation reaction, Fluor-de-Lys-Developer II (BioMol. Cat. #KI-176) was added to each well to digest the deacetylated substrate, thus producing the fluorescent signal. The reaction was allowed to develop for 45 minutes at 30 C. with 5% CO2; then the fluorescent signal was measured with an excitation wavelength at 360 nm. |
| Affinity data for this assay | |
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