Histone deacetylase (HDAC) enzymes act as transcriptional repressors of genes through the deacetylation of lysine residues on histone proteins. Sirtuins (SIRTs) are a unique class of deacetylases (class III HDACs) that require NAD as a cofactor. Sirtuin 1 (SIRT1) is involved in regulation of tumor suppressor p53 and inhibition of apoptosis. It also represents a target for treatment of age-related diseases and type II diabetes. The SensoLyte® Green SIRT1 Assay Kit provides a convenient, two-step homogeneous procedure for measuring Sirtuin 1 activity using a fluorogenic substrate. In the first step, an acetylated substrate is incubated with SIRT1 containing samples. Deacetylation of substrate first sensitizes it, so that, in the second step, mixing with the SIRT developer generates a fluorophore, which can then be detected at Ex/Em= 490 nm/520 nm. This kit can be used for high throughput screening of SIRT1 inhibitors/activators. The long wavelength fluorescence of SIRT1 substrate is less interfered by the autofluorescence of cell components and test compounds. Assays are performed in a standard 96-well format.
Component A: SIRT1 substrate: 1 mM, 50 mL
Component B: Deacetylated standard: 1 mM, 20 mL
Component C: SIRT1, Human Recombinant: 0.025 mg/mL, 400 mL
Component D: Assay Buffer: 20 mL
Component E: NAD+: 20 mM, 100 mL
Component F: Nicotinamide (Sirtuin Inhibitor): 30 mM, 0.5 mL
Component G: SIRT1 Developer (10X): 0.5 mL
Storage & stability
Store all kit components, except Component C, at -20°C. Store Component C at –80°C. Aliquot as needed to avoid freeze-thaw cycles. Protect Components A and B from light and from moisture. Component D can be stored at room temperature for convenience.