Recombinant Human Sirtuin 2/SIRT2 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Sirtuin 2/SIRT2 Protein, CF Summary

• Details of Functionality: Measured by its ability to remove the acetyl group from a fluorogenic peptide substrate Ac-RGK(Ac)-AMC (Catalog # ES016) in a coupled assay. The specific activity is >3 pmol/min/µg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Sirtuin 2/SIRT2 protein
  Met1-Gln389, with a C-terminal 6-His tag
• Accession #: Q8IXJ6
• N-terminal Sequence: No results obtained: Presumed blocked
• Protein/Peptide Type: Recombinant Enzymes
• Gene: SIRT2
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 44 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 43-49 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0
  • Stop Solution: 8 ng/µL Recombinant Human Active Trypsin 3/PRSS3 (Catalog # 3714-SE), 4 mM Nicotinamide (Sigma, Catalog # 72340), 50 mM Tris, 100 mM NaCl, 30% (v/v) isopropanol, pH 8.0
  • Recombinant Human Sirtuin 2/SIRT2 (rhSIRT2) (Catalog # 4358-DA)
  • beta -Nicotinamide adenine dinucleotide hydrate ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in diH₂O
  • Substrate: Ac-Arg-Gly-Lys(Ac)-AMC (Catalog # ES016), 20 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhSIRT2 to 0.04 µg/µL in Assay Buffer.
  2. Dilute Substrate to 500 µM in Assay Buffer containing 2 mM beta -NAD.
  3. Mix 25 µLof 0.04 µg/µL rhSIRT2 and 25 µL of Substrate in plate. Add 25 µL of 0.04 µg/µL rhSIRT2 alone for the control.
  4. Cover with parafilm or a plate sealer and incubate at 37 °C for 30 minutes.
  5. Add 50 µL of Stop Solution to all wells. Mix. For control well(s), add 25 µL of Substrate after addition of Stop Solution.
  6. Seal tightly and incubate at room temperature for 15 minutes.
  7. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in endpoint mode.
  8. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Control

  **Derived using calibration standard 7-amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

  Per Well:
  • rhSIRT2: 1 µg
  • Substrate: 125 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Sirtuin 2/SIRT2 Protein, CF

• EC 3.5.1
• EC 3.5.1.-
• FLJ35621
• FLJ37491
• S.cerevisiae, homolog) 2
• SIR2L2
• SIRT2
• Sirtuin 2
• sirtuin type 2

Background

Sirtuin 2, encoded by the SIRT2 gene, is also known as SIR2 (silent information regulator 2)-like protein 2. It is a nicotinamide adenine dinucleotide (NAD)-dependent histone/protein deacetylase (1, 2). The SIR2 family of enzymes is classified as class III histone deacetylases (HDACs) and has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging (3, 4). Unlike class I and class II HDACs, the enzymatic activity of class III HDACs is NAD dependent and insensitive to HDAC inhibitor trichostatin A (5).

1. Frye, R.A. (1999) Biochem. Biophys. Res. Commun. 260:273.
2. North, B.J. et al. (2003) Mol. Cell. 11:437.
3. North, B.J. and Verdin, E. (2004) Genome Biol. 5:224.
4. Marmorstein, R. (2004) Biochem. Soc. Trans. 32:904.
5. Gray, S.G. and Ekstrom, T.J. (2001) Exp. Cell Res. 262:75.  

Bioinformatics

• Gene Symbol: SIRT2
• Uniprot:
  • Q8IXJ6()