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FLUOR DE LYS® SIRT1 fluorometric drug discovery assay kit

First-to-market by the leader in Epigenetics research tools
 
BML-AK555-0001 96 wells 656.00 USD
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  • 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. The assay procedure has two steps. First, the FLUOR DE LYS® SIRT1 Substrate, which contains a peptide comprising amino acids 379-382 of human p53 (Arg-His-Lys-Lys(Ac)). Deacetylation of the substrate sensitizes the substrate so that, in the second step, treatment with the FLUOR DE LYS®Developer II produces a fluorophore.
bml-ak555

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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Product Specification

Alternative Name:Sirtuin 1 fluorescent assay kit
 
Applications:Fluorescent detection, HTS
Activity assay, Cell-based assays
 
Use/Stability:Store all components except the microplates and instruction booklet at -80°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 -80°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 -80°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.
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Kit/Set Contains:SIRT1 (Sirtuin 1, hSir2SIRT1) (human, recombinant) (Prod. No. BML-SE239)
(100 U; One U=1 pmol/min at 37°C, 250 µM, FLUOR DE LYS® Substrate (Prod. No. 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: -80°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: -80°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: -80°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: -80°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: -80°C
Resveratrol (Sirtuin Activator) (Prod. No. BML-KI284)
(10 mg; Solid MW: 228.2, soluble in DMSO or 100% ethanol (to 100mM)
Storage: -80°C
Suramin sodium (Sirtuin Inhibitor) (Prod. No. BML-KI285)
(10 mg; Solid MW: 1429.2, soluble in water or assay buffer (to 25mM))
Storage: -80°C
FLUOR DE LYS® Deacetylated Standard (Prod. No. BML-KI142)
(30 µl; 10mM in DMSO)
Storage: -80°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: -80°C
1/2 volume microplates (Prod. No. 80-2407)
1 clear and 1 white, 96-well
Storage: Room temperature
 
Scientific Background: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.
 
Technical Info/Product Notes:Cited example:
HTS application. Use of 384-well plates with this kit.
 
UniProt ID:Q96EB6
 

Product Literature References

Caspase-2 deficiency accelerates chemically induced liver cancer in mice: S. Shalini, et al.; Cell Death Differ. 23, 1727 (2016), Application(s): Measurement of liver metabolites and Sirt1 and Sirt3 activities, Abstract;
Conditioned Medium from Early-Outgrowth Bone Marrow Cells Is Retinal Protective in Experimental Model of Diabetes: D.A. Duarte, et al.; PLoS One 11, e0147978 (2016), Application(s): Measured SIRT1 activity, Abstract; Full Text
Dunnione ameliorates cisplatin ototoxicity through modulation of NAD+ metabolism: H.J. Kim, et al.; Hear Res. 333, 235 (2016), Application(s): SIRT1 activity Assay, Abstract;
SIRT1-Activating Compounds (STAC) Negatively Regulate Pancreatic Cancer Cell Growth and Viability Through a SIRT1 Lysosomal-Dependent Pathway: C.C. Chini et al.; Clin. Cancer Res. 22, 2496 (2016), Abstract;
SIRT1-AMPK crosstalk is involved in high glucose-dependent impairment of insulin responsiveness in primary rat podocytes: D. Rogacka, et al.; Exp. Cell. Res. 349, 328 (2016), Abstract;
SIRT3 Deacetylates Ceramide Synthases: Implications for Mitochondrial Dysfunction and Brain Injury: S.A. Novgorodov, et al.; J. Biol. Chem. 291, 1957 (2016), Application(s): Deacetylation assay with mouse brain cells, Abstract; Full Text
The peroxisome proliferator-activated receptor γ agonist pioglitazone prevents NF-κB activation in cisplatin nephrotoxicity through the reduction of p65 acetylation via the AMPK-SIRT1/p300 pathway: J. Zhang, et al.; Biochem. Pharmacol. 101, 100 (2016), Application(s): SIRT1 deacetylase activity, Abstract;
BET Inhibition Upregulates SIRT1 and Alleviates Inflammatory Responses: T. Kokkola, et al.; Chembiochem 16, 1997 (2015), Abstract; Full Text
Curcumin as therapeutics for the treatment of head and neck squamous cell carcinoma by activating SIRT1: A. Hu, et al.; Sci. Rep. 5, Article number 13429 (2015), Application(s): Deacetylase assay in HNSCC cell lines, Abstract; Full Text
Dunnione ameliorates cisplatin-induced small intestinal damage by modulating NAD+ metabolism: A. Pandit, et al.; Biochem. Biophys. Res. Commun. 467, 697 (2015), Application(s): Determination of SIRT1 activity, Abstract;
Leucine Amplifies the Effects of Metformin on Insulin Sensitivity and Glycemic Control in Diet-Induced Obese Mice: L. Fu, et al.; Metabolism 64, 845 (2015), Application(s): Assay, Abstract;
SIRT1 inhibition in pancreatic cancer models: Contrasting effects in vitro and in vivo: C. E. Oon, et al.; Eur. J. Pharmacol. 757, 59 (2015), Application(s): Fluorescence microscopy , Abstract;
Virtual screening approach of sirtuin inhibitors results in two new scaffolds: P. Kokkonen, et al.; Eur. J. Pharm. Sci. 76, 27 (2015), Application(s): Assay, Abstract;
A redox-resistant sirtuin-1 mutant protects against hepatic metabolic and oxidant stress: D. Shao, et al.; J. Biol. Chem. 289, 7293 (2014), Abstract;
Coumestrol induces mitochondrial biogenesis by activating Sirt1 in cultured skeletal muscle cells: D.B. Seo, et al.; J. Agric. Food Chem. 62, 4298 (2014), Abstract;
Design, synthesis, and biological activity of NCC149 derivatives as histone deacetylase 8-selective inhibitors: T. Suzuki, et al.; ChemMedChem. 9, 657 (2014), Abstract;
Modulation of the AMPK/Sirt1 axis during neuronal infection by herpes simplex virus type 1: C. Martin, et al.; J. Alzheimers Dis. 42, 301 (2014), Abstract;
Resveratrol delays Wallerian degeneration in a NAD(+) and DBC1 dependent manner: A. Calliari, et al.; Exp. Neurol. 251, 91 (2014), Abstract;
Synergistic Effects of Polyphenols and Methylxanthines with Leucine on AMPK/Sirtuin-Mediated Metabolism in Muscle Cells and Adipocytes: A. Bruckbauer & M.B. Zemel; PLoS One 9, e89166 (2014), Abstract; Full Text
Cytotoxicity and cell death mechanisms induced by a novel bisnaphthalimidopropyl derivative against the NCI-H460 non-small lung cancer cell line: R.T. Lima, et al.; Anticancer Agents Med. Chem. 13, 414 (2013), Abstract;
Dietary resveratrol prevents development of high-grade prostatic intraepithelial neoplastic lesions: involvement of SIRT1/S6K axis: G. Li, et al.; Cancer Prev. Res. (Phila) 6, 27 (2013), Abstract; Full Text
Effects of Resveratrol on the Recovery of Muscle Mass Following Disuse in the Plantaris Muscle of Aged Rats: B. Bennett, et al.; PLoS One 8, e83518 (2013), Abstract; Full Text
Modulation of p53 C-terminal acetylation by mdm2, p14ARF, and cytoplasmic SirT2: I.M. van Leeuwen, et al.; Mol. Cancer Ther. 12, 471 (2013), Abstract; Full Text
Resveratrol administration or SIRT1 overexpression does not increase LXR signaling and macrophage-to-feces reverse cholesterol transport in vivo: J.C. Escola-Gil, et al.; Transl. Res. 161, 110 (2013), Abstract;
Synergistic effects of metformin, resveratrol, and hydroxymethylbutyrate on insulin sensitivity: A. Bruckbauer & M.B. Zemel; Diabetes Metab. Syndr. Obes. 6, 93 (2013), Abstract; Full Text
Tenovin-D3, a novel small-molecule inhibitor of sirtuin SirT2, increases p21 (CDKN1A) expression in a p53-independent manner: A.R. McCarthy, et al.; Mol. Cancer Ther. 12, 352 (2013), Abstract; Full Text
A maternal high-fat diet modulates fetal SIRT1 histone and protein deacetylase activity in nonhuman primates: M.A. Suter, et al.; FASEB J. 26, 5106 (2012), Application(s): HDAC activity in fetal macaque liver or Cos-1 cell extracts, Abstract; Full Text
Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase: V. Nin, et al.; J. Biol. Chem. 287, 23489 (2012), Application(s): SIRT1 activity measurement in cells, Abstract; Full Text
SIRT1 contains N- and C-terminal regions that potentiate deacetylase activity: M. Pan, et al.; J. Biol. Chem. 287, 2468 (2012), Abstract; Full Text
Synergistic effects of leucine and resveratrol on insulin sensitivity and fat metabolism in adipocytes and mice: A. Bruckbauer, et al.; Nutr. Metab. (Lond.) 9, 77 (2012), Abstract; Full Text
Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila: C. Burnett, et al.; Nature 477, 482 (2011), Application(s): Drosophila's Sir2 deacetylation assay, Abstract; Full Text
Effects of dairy consumption on SIRT1 and mitochondrial biogenesis in adipocytes and muscle cells: A. Bruckbauer & M.B. Zemel; Nutr. Metab. (Lond.) 8, 91 (2011), Abstract; Full Text
Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation: A.A. Kendrick, et al.; Biochem. J. 433, 505 (2011), Application(s): SIRT1 activity in mouse liver cells, Abstract; Full Text
SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53: D.H. Kim, et al.; Am. J. Physiol. Renal Physiol. 301, F427 (2011), Application(s): Activity of immunoprecipitated SIRT1, Abstract; Full Text
SIRT inhibitors induce cell death and p53 acetylation through targeting both SIRT1 and SIRT2: B. Peck, et al.; Mol. Cancer Ther. 9, 844 (2010), Abstract; Full Text
Identification and characterization of novel sirtuin inhibitor scaffolds: B.D. Sanders, et al.; Bioorg. Med. Chem. 17, 7031 (2009), Application(s): HTS application. Use of 384-well plates with this kit, Abstract; Full Text

General Literature References

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

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