Recombinant Human Sirtuin 1/SIRT1 Protein, CF

R&D Systems | Catalog # 7714-DA

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human Sirtuin 1/SIRT1 Protein (7714-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

Q96EB6

Applications

Enzyme Activity
View all Sirtuin 1/SIRT1 Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Sirtuin 1/SIRT1 protein
Met1-Ser747, with a C-terminal 6-His tag.

Purity

>75%, 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

Ala2

Predicted Molecular Mass

82 kDa

SDS-PAGE

110-113 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 >15 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

7714-DA
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol.
Shipping The product is shipped with dry ice or equivalent. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: Sirtuin 1/SIRT1

SIRT1 is an NAD+-dependent class III histone deacetylase. It is a member of the sirtuin family of proteins, homologous to the yeast Sir2 protein. It modulates intracellular activities through transcription regulation and functions in several separate cellular functions. SIRT1 deacetylates and inactivates tumor suppressor protein p53, a process regulated by Hypermethylated in Cancer 1 (HIC1) protein to allow cells to bypass apoptosis and survive DNA damage (1, 2). The level of SIRT1 is significantly elevated in mouse and human prostate cancer (3). SIRT1 promotes vascular relaxation by activating endothelial nitric oxide synthase (4). SIRT1 also protects against microglia‑dependent amyloid-beta toxicity through inhibiting NF-Kappa b signaling (5).

References

  1. Langley, E. et al. (2002) EMBO J. 21:2383.
  2. Chen, W. Y. et al. (2005) Cell 123:437.
  3. Huffmun, D. M. et al. (2007) Cancer Res. 67:6612.
  4. Mattagajasingh, I. et al. (2007) Proc. Natl. Acad. Sci. 104:14855.
  5. Chen, J. et al. (2005) J. Biol. Chem. 280:40364.

Long Name

Silent Mating Type Information Regulation 2 Homolog

Alternate Names

SIR2-like protein 1, SIR2alpha, SIR2L1, SIRT1

Entrez Gene IDs

23411 (Human); 93759 (Mouse); 309757 (Rat)

Gene Symbol

SIRT1

UniProt

Q96EB6 (Human)

Additional Sirtuin 1/SIRT1 Products

Product Documents for Recombinant Human Sirtuin 1/SIRT1 Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human Sirtuin 1/SIRT1 Protein, CF

For research use only

Citations for Recombinant Human Sirtuin 1/SIRT1 Protein, CF

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Protocols

View specific protocols for Recombinant Human Sirtuin 1/SIRT1 Protein, CF (7714-DA):

Materials
  • Assay Buffer: 50 mM Tris, 1 mM DTT, pH 9.0
  • Stop Buffer: 50 mM Tris, 100 mM NaCl, 30% (v/v) isopropanol, pH 8.0
  • Recombinant Human Sirtuin 1/SIRT1 (rhSIRT1) (Catalog # 7714-DA)
  • Fluorogenic Peptide Substrate Ac-Arg-Gly-Lys(Ac)-AMC (Catalog # ES016)
  • beta -nicotinamide adenine dinucleotide hydrate ( beta -NAD) (Sigma, Catalog # N6522), 100 mM in diH2O
  • Recombinant Mouse Active Trypsin 3/PRSS3 (Trypsin 3) (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 rhSIRT1 to 10 µg/mL in Assay Buffer.
  2. Dilute Substrate to 500 µM in Assay Buffer containing 2 mM beta -NAD.
  3. Load 25 µL of 10 µg/mL rhSIRT1 into the plate in triplicate and start the reaction by adding 25 µL of 500 µM Substrate. Include a control containing 25 µL of 10 ng/µL rhSIRT1 only.
  4. Incubate sealed plate at 37 °C for 30 minutes.
  5. Prepare Stop Solution by combining Trypsin 3 and Nicotinamide in Stop Buffer to final concentrations of 0.2 µg/mL and 4 mM, respectively.
  6. Add 50 µL of Stop Solution to all wells. Add 25 µL of 500 µM Substrate to the enzyme control wells after the 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.

     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).

Per Well:

  • rhSIRT1: 0.25 µg
  • Substrate: 125 µM

FAQs

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