Recombinant Human Sirtuin 1/SIRT1 Protein, CF Summary
Product Specifications
Met1-Ser747, with a C-terminal 6-His tag.
Analysis
Product Datasheets
7714-DA
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
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.
|
Assay Procedure
- 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
- Dilute rhSIRT1 to 10 µg/mL in Assay Buffer.
- Dilute Substrate to 500 µM in Assay Buffer containing 2 mM beta -NAD.
- 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.
- Incubate sealed plate at 37 °C for 30 minutes.
- Prepare Stop Solution by combining Trypsin 3 and Nicotinamide in Stop Buffer to final concentrations of 0.2 µg/mL and 4 mM, respectively.
- 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.
- Seal plate tightly with plate sealer and incubate at room temperature for 15 minutes.
- 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).
- 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).
- Langley, E. et al. (2002) EMBO J. 21:2383.
- Chen, W. Y. et al. (2005) Cell 123:437.
- Huffmun, D. M. et al. (2007) Cancer Res. 67:6612.
- Mattagajasingh, I. et al. (2007) Proc. Natl. Acad. Sci. 104:14855.
- Chen, J. et al. (2005) J. Biol. Chem. 280:40364.
Citations for Recombinant Human Sirtuin 1/SIRT1 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Sirtuin 1-mediated deacetylation of XPA DNA repair protein enhances its interaction with ATR protein and promotes cAMP-induced DNA repair of UV damage
Authors: SG Jarrett, KM Carter, RM Bautista, D He, C Wang, JA D'Orazio
J. Biol. Chem., 2018-10-16;0(0):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Acetylation of Mammalian ADA3 is Required for its Functional Roles in Histone Acetylation and Cell Proliferation
Mol Cell Biol, 2016-09-12;0(0):.
Species: Human
Sample Types: Protein
Applications: Bioassay
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