Recombinant Human Sirtuin 3/SIRT3 Protein, CF

Catalog #: 7488-DA
1 Citation Datasheet / COA / SDS

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7488-DA-050

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Recombinant Human Sirtuin 3/SIRT3 Protein, CF Summary

Product Specifications

Purity

>95%, 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.

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 >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 #

Q9NTG7

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

34 kDa

SDS-PAGE

30-33 kDa, reducing conditions

Product Datasheets

7488-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.

7488-DA

Formulation	Supplied as a 0.2 μm filtered solution in Tris, NaCl, Brij-35 and Glycerol.
Shipping	The product is shipped with polar packs. 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, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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

Dilute rhSIRT3 to 40 ng/µL in Assay Buffer.
Dilute Substrate to 1 mM in Assay Buffer containing 2 mM beta -NAD.
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.
Seal plate and incubate at 37 °C for 30 minutes.
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.
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.
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.
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

Reconstitution Calculator

Background: Sirtuin 3/SIRT3

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).

References

North, B. J. et al. (2004) Genome Biology 5:224.
Schwer, B. et al. (2006) Proc. Natl. Acad. Sci. 103:11224.
Schlicker, C. et al. (2008) J. Mol. Biol. 382:790.
Ahn, B.-H. et al. (2008) Proc. Natl. Acad. Sci. 105:14447.
Jin, L. et al. (2009) J. Biol. Chem. 284: 24394.
Rose, G. et al. (2003) Exp. Gerontol. 38: 1065.

Long Name

Silent Mating Type Information Regulation 3 Homolog

Entrez Gene IDs

23410 (Human); 64384 (Mouse); 293615 (Rat)

Alternate Names

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

Related Research Areas

Citation for Recombinant Human Sirtuin 3/SIRT3 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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