Recombinant Human Sirtuin 3/SIRT3 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human Sirtuin 3/SIRT3 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 >2 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human Sirtuin 3/SIRT3 protein
Ser101-Lys399, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Met
Protein/Peptide Type
Recombinant Enzymes
Gene
SIRT3
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

Theoretical MW
34 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
30-33 kDa, reducing conditions
Publications
Read Publication using
7488-DA in the following applications:

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, NaCl, Brij-35 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 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

Notes

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

Alternate Names for Recombinant Human Sirtuin 3/SIRT3 Protein, CF

  • EC 3.5.1
  • EC 3.5.1.-
  • hSIRT3
  • mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase
  • NAD-dependent deacetylase sirtuin-3, mitochondrial
  • SIR2L3
  • SIR2L3silent mating type information regulation 2, S.cerevisiae, homolog 3
  • sir2-like 3
  • SIR2-like protein 3
  • SIRT3
  • sirtuin (silent mating type information regulation 2 homolog) 3 (S. cerevisiae)
  • sirtuin (silent mating type information regulation 2, S.cerevisiae, homolog) 3
  • Sirtuin 3
  • sirtuin type 3

Background

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).
  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.

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Publications for Sirtuin 3/SIRT3 (7488-DA)(1)

We have publications tested in 1 confirmed species: Bovine.

We have publications tested in 1 application: Bioassay.


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Bioinformatics

Gene Symbol SIRT3
Uniprot