The Fluorogenic SIRT1 Assay Kit is a complete assay system designed to measure Sirtuin 1 (SIRT1) activity for screening and profiling applications. It comes in a convenient 96-well format, with all the reagents necessary for 100 fluorescent SIRT1 (Sir2) activity measurements. In addition, the kit includes purified SIRT1 enzyme and a SIRT inhibitor, Nicotinamide, for use as a positive and negative control, respectively. The Fluorogenic SIRT1 Assay Kit is based on a unique fluorogenic substrate and developer combination. This assay method eliminates dealing with the radioactivity, extraction, and chromatography aspects of traditional assays. Using this kit, only two simple steps on a microtiter plate are needed to analyze the SIRT1 activity level. First, the SIRT fluorometric substrate (SIRT substrate 1), containing an acetylated lysine side chain, is incubated with a sample containing SIRT1 activity (e.g. HeLa nuclear extract or purified SIRT1 enzyme). The deacetylation sensitizes the substrate so subsequent treatment with the Lysine Developer produces a fluorophore that can then be measured using a fluorescence reader. Intended Use: Designed to measure Sirtuin 1 (SIRT1) activity for screening and profiling applications. Test Principle: The Fluorogenic SIRT1 Assay Kit is based on a unique fluorogenic substrate and developer combination. This assay method eliminates dealing with the radioactivity, extraction, and chromatography aspects of traditional assays. Using this kit, only two simple steps on a microtiter plate are needed to analyze the SIRT1 activity level. First, the SIRT fluorometric substrate (SIRT substrate 1), containing an acetylated lysine side chain, is incubated with a sample containing SIRT1 activity (e.g. HeLa nuclear extract or purified SIRT1 enzyme). The deacetylation sensitizes the substrate so subsequent treatment with the Lysine Developer produces a fluorophore that can then be measured using a fluorescence reader. Kit Components: SIRT1 recombinant human enzyme: 1x50ug (0.06mg/ml) Fluorogenic SIRT substrate 1: 1x50ul 5mM Nicotinamide Adenine Dinucleotide (NAD+): 1x50ul 50mM Nicotinamide: 1x500ul 10mM 2x SIRT Developer (contains 2 mM Nicotinamide): 1x6ml Trichostatin A (200uM) 1x100ul SIRT assay buffer: 1x10ml Black, low binding NUNC black microtiter plate: 1 plate Storage and Stability: Store other components at 4C. Stable for 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
