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FLUOR DE LYS^® SIRT2 fluorometric drug discovery assay kit

BML-AK556

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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 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.

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.

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FLUOR DE LYS® SIRT2 fluorometric drug discovery assay kit image — **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.

Product Details

Alternative Name	Sirtuin 2 fluorescent assay kit
Application	Activity assay, Cell-based assays, Fluorescent detection, HTS
Contents	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
UniProt ID	Q8IXJ6

Handling & Storage

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.
Long Term Storage	-80°C
Shipping	Dry Ice

Regulatory Status	RUO – Research Use Only

Novel inhibitors of bromodomain and extra-terminal domain trigger cell death in breast cancer cell lines: M. Rahnasto-Rilla, et al.; Bioorg. Med. Chem. 112, 117884 (2024), Abstract
Structure-Guided Design of a Small-Molecule Activator of Sirtuin-3 that Modulates Autophagy in Triple Negative Breast Cancer: J. Zhang, et al.; J. Med. Chem. 64, 14192 (2021), Abstract
Design and in vitro analysis of SIRT2 inhibitor targeting Parkinson’s disease: A.P. Singh, et al.; Mol. Divers. 25, 2261 (2021), Abstract
Pharmacophore modeling and virtual screening studies to identify novel selective SIRT2 inhibitors: G. Eren, et al.; J. Mol. Graph. Model. 10, 1313 (2019), Abstract
Sirtuin 2 mutations in human cancers impair its function in genome maintenance: P.E. Head, et al.; J. Biol. Chem. 292, 9919 (2017), Abstract — Full Text
Aquatide Activation of SIRT1 Reduces Cellular Senescence through a SIRT1-FOXO1-Autophagy Axis: C.J. Lim, et al.; Biomol. Ther. 25, 511 (2017), Abstract — Full Text
Discovery of bicyclic pyrazoles as class III histone deacetylase SIRT1 and SIRT2 inhibitors: E. Therrien, et al.; Bioorg. Med. Chem. Lett. 25, 2514 (2015), Abstract
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

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FLUOR DE LYS^® SIRT2 fluorometric drug discovery assay kit

BML-AK556

Product Details

Handling & Storage

Datasheet, Manuals, SDS & CofA

Manuals And Inserts

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