Recombinant Human Sirtuin 1/SIRT1 Protein, CF

Catalog #: 7714-DA
2 Citations Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
7714-DA-050

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human Sirtuin 1/SIRT1 Protein, CF Summary

Product Specifications

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 # ES016) in a coupled assay. The specific activity is >15 pmol/min/μg, as measured under the described conditions.

Source

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

Accession #

Q96EB6

N-terminal Sequence
Analysis

Ala2

Predicted Molecular Mass

82 kDa

SDS-PAGE

110-113 kDa, reducing conditions

Product Datasheets

7714-DA

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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. 6 months from date of receipt, -70 °C as supplied. 3 months, -70 °C under sterile conditions after opening.

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 # 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).

Per Well:

rhSIRT1: 0.25 µg
Substrate: 125 µM

Reconstitution Calculator

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

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.

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

Related Research Areas

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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