Recombinant Human Sirtuin 3/SIRT3 Protein, CF

R&D Systems | Catalog # 7488-DA

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human Sirtuin 3/SIRT3 Protein (7488-DA)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

Q9NTG7

Applications

Enzyme Activity
View all Sirtuin 3/SIRT3 Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Sirtuin 3/SIRT3 protein
Ser101-Lys399, with an N-terminal Met and 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

34 kDa

SDS-PAGE

30-33 kDa, reducing conditions

Activity

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 >2 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

7488-DA
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Brij-35 and Glycerol.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Background: Sirtuin 3/SIRT3

Sirtuin 3 (SIRT3) is a NAD+-dependent class III histone deacetylase. It is primarily compartmentalized to mitochondria (1) and activates mitochondrial target proteins, including ACSS1, IDH2 and GDH by deacetylating key lysine residues (2-4). SIRT3 plays an important role in regulating mitochondrial metabolism and energy production and thus has emerged as a potential therapeutic target to treat metabolic and neurological diseases (5). Polymorphisms in human SIRT3 have been linked to survivorship among the elderly, suggesting a possible involvement of SIRT3 in age-related phenomena (6).

References

  1. North, B. J. et al. (2004) Genome Biology 5:224.
  2. Schwer, B. et al. (2006) Proc. Natl. Acad. Sci. 103:11224.
  3. Schlicker, C. et al. (2008) J. Mol. Biol. 382:790.
  4. Ahn, B.-H. et al. (2008) Proc. Natl. Acad. Sci. 105:14447.
  5. Jin, L. et al. (2009) J. Biol. Chem. 284: 24394.
  6. Rose, G. et al. (2003) Exp. Gerontol. 38: 1065.

Long Name

Silent Mating Type Information Regulation 3 Homolog

Alternate Names

SIR2-like protein 3, SIR2L3, SIRT3

Entrez Gene IDs

23410 (Human); 64384 (Mouse); 293615 (Rat)

Gene Symbol

SIRT3

UniProt

Q9NTG7 (Human)

Additional Sirtuin 3/SIRT3 Products

Product Documents for Recombinant Human Sirtuin 3/SIRT3 Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human Sirtuin 3/SIRT3 Protein, CF

For research use only

Related Research Areas

Epigenetics

Citations for Recombinant Human Sirtuin 3/SIRT3 Protein, CF

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Protocols

View specific protocols for Recombinant Human Sirtuin 3/SIRT3 Protein, CF (7488-DA):

Materials
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 9.0
  • Stop Buffer: 50 mM Tris, 100 mM NaCl, 30% (v/v) Isopropanol, pH 8.0
  • Recombinant Human Sirtuin 3/SIRT3 (rhSIRT3) (Catalog # 7488-DA)
  • Fluorogenic Peptide Substrate Ac-Arg-Gly-Lys(Ac)-AMC  (Catalog # ES016), 20 mM in DMSO
  • beta -nicotinamide adenine dinucleotide hydrate ( beta -NAD) (Sigma, Catalog # N6522, 100 mM in diH2O)
  • Recombinant Mouse Active Trypsin 3/PRSS3 (Catalog # 7235-SE)
  • Nicotinamide (Sigma, Catalog # 72340, 100 mM in diH2O)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhSIRT3 to 40 ng/µL in Assay Buffer.
  2. Dilute Substrate to 1 mM in Assay Buffer containing 2 mM beta -NAD.
  3. Load into plate 25 µL of 40 ng/µL rhSIRT3 in triplicate and start the reaction by adding 25 µL of 1 mM Substrate. As a control, load 25 µL of 40 ng/µL rhSIRT3 in triplicate.
  4. Seal plate and incubate at 37 °C for 30 minutes.
  5. Prepare Stop Solution by combining Recombinant Mouse Trypsin 3/PRSS3 and Nicotinamide in Stop Buffer to final concentrations of 0.2 ng/µL and 4 mM, respectively.
  6. Add 50 µL of Stop Solution to all wells. To the control wells, add 25 µL of 1 mM Substrate mixture after addition of Stop Solution.
  7. Seal plate tightly with plate sealer and incubate at room temperature for 15 minutes.
  8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
  9. 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 # A9801).

  • rhSIRT3: 1.0 µg
  • Substrate: 250 µM

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