• Purity

  >75%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane.

• Endotoxin Level

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

• Activity

  Measured by its ability to remove the acetyl group from a fluorogenic peptide substrate Ac-RGK(Ac)-AMC (Catalog # ) in a coupled assay. The specific activity is >15 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Met1-Ser747, with a C-terminal 6-His tag.

• Accession #

• N-terminal Sequence    
  Analysis

  Ala2

• Predicted Molecular Mass

  82 kDa

• SDS-PAGE

  110-113 kDa, reducing conditions

Assay Procedure

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 # )

• beta -nicotinamide adenine dinucleotide hydrate ( beta -NAD) (Sigma, Catalog # N6522), 100 mM in diH₂O

• Recombinant Mouse Active Trypsin 3/PRSS3 (Trypsin 3) (Catalog # )

• 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.

     *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

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

• Entrez Gene IDs:

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

• Alternate Names:

  EC 3.5.1; S. cerevisiae, homolog) 1; SIR2alpha; SIR2L1; SIR2-like protein 1; SIRT1; Sirtuin 1