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FLUOR DE LYS® SIRT2 fluorometric drug discovery assay kit

 
BML-AK556-0001 96 wells 656.00 USD
Do you need bulk/larger quantities?
 
  • 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 SIRT2 Fluorescent Activity Assay/Drug Discovery Kit is a complete assay system designed to measure the lysyl deacetylase activity of the recombinant human SIRT2 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® SIRT2 assay is based on the FLUOR DE LYS®Substrate and FLUOR DE LYS® Developer combination. The assay procedure has two steps. First, the FLUOR DE LYS® SIRT2 Substrate, which comprises a unique peptide based on amino acids 317-320 of p53 (Gln-Pro-Lys-Lys(Ac)), is incubated with SIRT2. 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-ak556
Figure 1: Reaction Scheme of the SIRT2 Fluorescent Activity Assay. NAD+-dependent deacetylation of the substrate by recombinant human SIRT2 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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bml-ak556

Product Specification

Alternative Name:Sirtuin 2 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 SIRT2 enzyme, Prod. No. BML-SE251, 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 extract 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:SIRT2 (Sirtuin 2, hSir2) (human, recombinant) (Prod. No. BML-SE25)
(500 U, one U=1 pmol/min at 37°C, 500µM, FLUOR DE LYS®-SIRT2 substrate (Prod. No. BML-KI179), 500µM 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®-SIRT2, Deacetylase substrate (Prod. No. BML-KI179)
(100µl; 5mM solution in 25mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassion chloride, 1mM magnesium chloride)
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; 5 mM Nicotinamide in 50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride)
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 (Prod. No. BML-KI286)
(20 ml; 50mM TRIS/Cl, pH 8.0, 137mM sodium chloride, 2.7mM potassium chloride, 1mM magnesium chloride, 1 mg/ml bovine serum albumin)
Storage: -80°C
1/2 volume microplate (Prod. No. BML-KI101)
Storage: Room temperature
1/2 volume white microplate (Prod. No. BML-KI110)
Storage: Room temperature
 
Scientific Background:Due to their roles in gene silencing, aging and oxidative stress responses, the sirtuins (NAD -dependent deacetylases related to yeast Sir2), are currently the subject of intense research interest. Like Sir2, human SIRT2 is a class I sirtuin, although not as closely related to Sir2 as human SIRT1. The three-dimensional structure of SIRT2 has been determined, and its catalytic core has the same basic two-domain architecture and central groove as other sirtuins. SIRT2 is a cytoplasmic protein, although a lesser amount of nuclear localization has also been reported and an interaction with the homeobox transcription factor HOXA10 have also been reported. A complex comprising HDAC6 and SIRT2 colocalizes with the microtubule network and both enzymes can deacetylate a-tubulin lysine-40 in purified tubulin heterodimers or microtubules. SIRT2 protein levels rise and it becomes phosphorylated during mitosis . Overexpression of SIRT2 delays exit of cells from mitosis, suggesting that it may play a role in cell cycle regulation.
 
UniProt ID:Q8IXJ6
 

Product Literature References

Design, synthesis and structure-activity relationship studies of novel sirtuin 2 (SIRT2) inhibitors with a benzamide skeleton: T. Sakai, et al.; Bioorg. Med. Chem. 23, 328 (2015), 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;
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;
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
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
Discovery and validation of SIRT2 inhibitors based on tenovin-6: use of a ¹H-NMR method to assess deacetylase activity: L. Pirrie, et al.; Molecules 17, 12206 (2012), Abstract; Full Text
Design of a novel nucleoside analog as potent inhibitor of the NAD+ dependent deacetylase, SIRT2: P. Sivaraman, et al.; Syst. Synth. Biol. 4, 257 (2010), 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

General Literature References

Human histone deacetylase SIRT2 interacts with the homeobox transcription factor HOXA10: N.S. Bae, et al.; J. Biochem. 135, 695 (2004), Abstract;
Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli: K. Zhao, et al.; J. Mol. Biol. 337, 731 (2004), Abstract;
Role for human SIRT2 NAD-dependent deacetylase activity in control of mitotic exit in the cell cycle: S.C. Dryden, et al.; Mol. Cell Biol. 23, 3173 (2003), Abstract;
Structure and autoregulation of the yeast Hst2 homolog of Sir2: K. Zhao, et al.; Nat. Struct. Biol. 10, 864 (2003), Abstract;
Structure of the yeast Hst2 protein deacetylase in ternary complex with 2’-O-acetyl ADP ribose and histone peptide: K. Zhao, et al.; Structure 11, 1403 (2003), Abstract;
The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase: J. North, et al.; Mol. Cell 11, 437 (2003), Abstract;
Structure of a Sir2 enzyme bound to an acetylated p53 peptide: J.L. Avalos, et al.; Mol. Cell 10, 523 (2002), 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 (2001), Abstract;
Crystal structure of a SIR2 homolog-NAD complex: J. Min, et al.; Cell 105, 269 (2001), Abstract;
Structure of the histone deacetylase SIRT2: M.S. Finnin, et al.; Nat. Struct. Biol. 8, 621 (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;
Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins: R.A. Frye, et al.; Biochem. Biophys. Res. Commun. 273, 793 (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), Abstract;
Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2: G. Afhsar, et al.; Gene 234, 161 (1999), 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;
Silencers, silencing, and heritable transcriptional states: P. Laurenson, et al.; Microbiol. Rev. 56, 543 (1992), Abstract;

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