Fluor-de-Lys^® SIRT1 fluorometric drug discovery assay kit

[Sirtuin 1 fluorescent assay kit]



BML-AK555-0001	1 Kit	528.00 USD

Useful for inhibitor screening or characterizing enzyme kinetics
Includes optimal substrate selected from a panel of acetylated sites in p53 and histones
Supplied with enough recombinant enzyme for 96 assays (1 x 96-well plate)

A Fluor de Lys^® fluorescent assay system. The SIRT1 Fluorescent Activity Assay/Drug Discovery Kit is a complete assay system designed to measure the lysyl deacetylase activity of the recombinant human SIRT1 included in the kit. The kit is ideal for chemical library screening for candidate inhibitors or activators or kinetic assay of the enzyme under varying conditions. The Fluor de Lys^® SIRT1 assay is based on the Fluor de Lys^®SIRT1 Substrate and Fluor de Lys^® Developer II combination.

Quantity:	96 assays

Kit/Set Contains:	SIRT1 (Sirtuin 1, hSir2^SIRT1) (human, recombinant) (Prod. No. BML-SE239) (100 U; One U=1 pmol/min at 37°C, 250 µM, Fluor de Lys^® Substrate (BML-KI104), 500 µM NAD ; Recombinant enzyme dissolved in 25mM TRIS, pH 7.5, 100mM sodium chloride, 5mM dithiothreitol and 10% glycerol. See vial label for activity and protein concentrations) Storage: -70°C; AVOID FREEZE/THAW CYCLES! Fluor de Lys^® SIRT1, Deacetylase Substrate (Prod. No. BML-KI177) (100µl; 5mM solution in 50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride) Storage: -70°C Fluor de Lys^® Developer II Concentrate (5x) (Prod. No. BML-KI176) (5 x 250 µl; 5x Stock Solution; Dilute in Assay Buffer before use) Storage: -70°C NAD (Sirtuin Substrate) (Prod. No. BML-KI282) (500 µl; 50 mM β-Nicotinamide adenine dinucleotide (oxidized form) in 50mM TRIS/CL, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride) storage: -70°C Nicotinamide (Sirtuin Inhibitor) (Prod. No. BML-KI283) (500µl; 50 mM Nicotinamide in 50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride) Storage: -70°C Resveratrol (Sirtuin Activator) (Prod. No. BML-KI284) (10 mg; Solid MW: 228.2, soluble in DMSO or 100% ethanol (to 100mM) Storage: -70°C Suramin sodium (Sirtuin Inhibitor) (Prod. No. BML-KI285) (10 mg; Solid MW: 1429.2, soluble in water or assay buffer (to 25mM)) Storage: -70°C Fluor de Lys^® Deacetylated Standard (Prod. No. BML-KI142) (30 µl; 10mM in DMSO) Storage: -70°C Sirtuin Assay Buffer (50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride, 1 mg/ml bovine serum albumin) (Prod. No. BML-KI286) (20 ml)Storage: -70°C 1/2 volume microplate (Prod. No. BML-KI101) Storage: Room temperature 1/2 volume white microplate (Prod. No. BML-KI571) Storage: Room temperature

Shipping:	SHIPPED ON DRY ICE

Long Term Storage:	-80°C

Use/Stability:	Store all components except the microplates and instruction booklet at -70°C for the highest stability. The SIRT1 enzyme, (Prod. No. BML-SE239), must be handled with particular care in order to retain maximum enzymatic activity. Defrost it quickly in a RT water bath or by rubbing between fingers, then immediately store on an ice bath. The remaining unused enzyme should be refrozen quickly, by placing at -70°C. If possible, snap freeze in liquid nitrogen or a dry ice/ethanol bath. To minimize the number of freeze/thaw cycles, aliquot into separate tubes and store at -70°C. The 5x Developer II (Prod. No. BML-KI176) can be prone to precipitation if thawed too slowly. It is best to thaw this reagent in a room temperature water bath and, once thawed, transfer immediately onto ice.

Miscellaneous/General:	Yeast Sir2 (Silent information regulator 2) is the founding exemplar of the ’sirtuins’, an apparently ancient group of enzymes that occurs in eukaryotes, the archaea and eubacteria. In yeast and C. elegans, added copies of sirtuin genes extend lifespan and Sir2 is required for the lifespan extension conferred by caloric restriction in yeast. There are seven human sirtuins, which have been designated SIRT1-SIRT7. SIRT1, which is located in the nucleus, is the human sirtuin with the greatest homology to Sir2 and has been shown to exert a regulatory effect on p53 by deacetylation of lysine-382. Dr. Konrad Howitz at Enzo Life Sciences carried out a screen for modulators of SIRT1 activity which yielded a number of small molecule activators, all of which were plant polyphenols. Several of these Sirtuin Activating Compounds (STACs) extended yeast lifespan in a way that mimicked caloric restriction. Resveratrol, the most potent of these STACs activated SIRT1 in human cells and enhanced the survival rate of cells stressed by irradiation.

Background / Technical Information:	Please click here for the comprehensive product datasheet.

Figure: Reaction Scheme of the SIRT1 Fluorescent Activity Assay*. NAD+-dependent deacetylation of the substrate by recombinant human SIRT1 sensitizes it to Developer II, which then generates a fluorophore (symbol). The fluorophore is excited with 360 nm light and the emitted light (460 nm) is detected on a fluorometric plate reader. NAD+ is consumed in the reaction to produce nicotinamide (NAM) and O-acetyl-ADP-ribose.

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General Literature References

Silencers, silencing, and heritable transcriptional states P. Laurenson et al. Microbiol Rev 56 543 (1992) Abstract

The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms M. Kaeberlein et al. Genes Dev. 13 2570 (1999) Abstract

A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family J. S. Smith et al. PNAS 97 6658 (2000) Abstract

Caloric restriction and aging: an update E.J. Masoro et al. Exp. Gerontol. 35 299 (2000) Abstract

Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product J. C. Tanny et al. PNAS 98 415 (2000) Abstract

Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins R. A. Frye et al. Biochem. Biophys. Res. Commun. 273 793 (2000) Abstract

Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae S.J. Lin et al. Science 289 2126 (2000) Abstract

Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose K. G. Tanner et al. PNAS 97 14178 (2000) Abstract

Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase S. Imai et al. Nature 403 795 (2000)

hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase H. Vaziri et al. Cell 107 149 (2001) Abstract

Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans H. A. Tissenbaum et al. Nature 410 227 (2001) Abstract

Negative control of p53 by Sir2alpha promotes cell survival under stress J. Luo et al. Cell 107 137 (2001) Abstract

Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence E. Langley et al. Embo J. 21 2383 (2002) Abstract

Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1 K.J. Bitterman et al. J. Biol. Chem. 277 45099 (2002) Abstract

Calorie restriction in rhesus monkeys J.A. Mattison Exp. Gerontol. 38 35 (2003) Abstract

Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae R. M. Anderson et al. Nature 423 181 (2003) Abstract

Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan K. T. Howitz et al. Nature 425 191 (2003) Abstract