Recombinant Human Sirtuin 5/SIRT5 His-tag Protein, CF

R&D Systems | Catalog # 10270-DA

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Key Product Details

  • R&D Systems Sf 21 (baculovirus)-derived Recombinant Human Sirtuin 5/SIRT5 His-tag Protein (10270-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

Sf 21 (baculovirus)

Accession Number

Q9NXA8

Applications

Enzyme Activity
View all Sirtuin 5/SIRT5 Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Sirtuin 5/SIRT5 protein
Met33-Ser310
with a C-terminal 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met33 & Ser37

Predicted Molecular Mass

31 kDa

SDS-PAGE

33 kDa under reducing conditions

Activity

Measured by its ability to remove the succinyl group from a fluorogenic peptide substrate in a coupled assay.
The specific activity is >20 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant Human Sirtuin 5/SIRT5 His-tag Protein, CF

Recombinant Human Sirtuin 5/SIRT5 His-tag Protein SDS-PAGE

Recombinant Human Sirtuin 5/SIRT5 His-tag Protein SDS-PAGE

2 μg/lane of Recombinant Human Sirtuin 5/SIRT5 His-tag was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at 33 kDa under reducing conditions.

Formulation, Preparation, and Storage

10270-DA
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
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, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: Sirtuin 5/SIRT5

Sirtuin 5, encoded by the SIRT5 gene, is a nicotinamide adenine dinucleotide (NAD)-dependent enzyme that is part of the SIRT family also known as the SIR2 (silent information regulator 2)-like protein family.  The SIR2 family of enzymes is classified as class III histone deacetylases (HDACs). The family is comprised of seven family members, SIRT1-7 that contain the conserved NAD-binding and catalytic domains but have distinct N- and C-termini that contribute differences in specificity, subcellular localization and enzymatic activity (1,2). SIRT5 is a 310 amino acid protein with broad tissue distribution (3,4) and primarily localized to the mitochondrial matrix (3,4) with a larger acyl binding pocket and unique active site residues compared to other SIRTs (1,2). SIRT5 possesses very weak deacetylase activity but can perform protein desuccinylation, demalonylation, and deglutarylation (1,2) due to its altered binding pocket. It is the principal regulator of these modifications in mitochondrial as well as the cytosolic and nuclear proteins based on proteomic detection of hundreds of SIRT5 substrate molecules (5-9). SIRT5 is implicated to impact cellular processes including metabolism (9-11) and fatty acid B-oxidation (6,11,12) in maintenance of cardiac homeostasis (12,13), cancer (14-16), and neurodegenerative disorders (17,18). Given its role in many pathologies, there is significant interest in targeting SIRT5 therapeutically (11,16).

References

  1. Peng, C. et al. (2011) Mol. Cell Proteomics 10:M111.012658.
  2. Du, J. et al. (2011) Science. 334:806.
  3. Michishita, E. et al. (2005) Mol. Biol. Cell 16:4623.
  4. Nakagawa, T. et al. (2009) Cell 137:560.
  5. Park, J. et al. (2013) Mol. Cell 50:919.
  6. Rardin, M.J. et al. (2013) Cell Metab. 18:920.
  7. Tan, M. et al. (2014) Cell Metab. 19:605.
  8. Colak, G. et al. (2015) Mol. Cell Proteomics 14:3056.
  9. Nishida, Y. et al. (2015) Mol. Cell 59:321.
  10. Wang, F. et al. (2017) Cell Rep. 19:2331.
  11. Kumar, S and D. B. Lombard. (2018) Crit. Rev. Biochem. Mol. Biol. 53:311.
  12. Sadhukhan, S. et al. (2016) Proc. Natl. Acad. Sci. USA 113:4320.
  13. Hershberger, K.A. et al. (2017) J. Biol. Chem. 292:19767.
  14. Lu, W. et al. (2014) Tumour Biol. 35:10699.
  15. Zhao, T. et al. (2017) Oncotarget 8:53819.
  16. Bringman-Rodenbarger, L.R. et al. (2018) Antioxid. Redox Signal 28:677.
  17. Li, F and L. Liu. (2016) Front. Cell Neurosci. 10:171.
  18. Koronowski, K.B. et al. (2018) Front. Neurosci. 12:32.

Long Name

Silent Mating Type Information Regulation 5 Homolog

Alternate Names

SIR2L5, SIRT5

Entrez Gene IDs

23408 (Human); 68346 (Mouse); 306840 (Rat)

Gene Symbol

SIRT5

UniProt

Q9NXA8

Additional Sirtuin 5/SIRT5 Products

Product Documents for Recombinant Human Sirtuin 5/SIRT5 His-tag Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human Sirtuin 5/SIRT5 His-tag Protein, CF

For research use only

Related Research Areas

Epigenetics

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Protocols

View specific protocols for Recombinant Human Sirtuin 5/SIRT5 His-tag Protein, CF (10270-DA):

Materials
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0
  • Stop Solution: 8 ng/µL Recombinant Human Active Trypsin 3/PRSS3 (Catalog # 3714-SE), 4 mM Nicotinamide (Sigma, Catalog # 72340), 50 mM Tris, 100 mM NaCl, 30% (v/v) isopropanol, pH 8.0
  • Recombinant Human Sirtuin 5/SIRT5 (rhSIRT5) (Catalog # 10270-DA)
  • Nicotinamide adenine dinucleotide sodium salt ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in diH2O
  • Substrate: FLUOR DE LYS®-Succinyl, Desuccinylase Substrate (5 mM) (Enzo Life Sciences Int., Catalog # BML-KI590)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhSIRT5 to 1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 100 µM in Assay Buffer containing 2 mM beta -NAD.
  3. Combine 25 µL of 1 ng/µL rhSIRT5 and 25 µL of dilute Substrate in plate. For Control, load 25 µL of 1 ng/µL rhSIRT5 alone.
  4. Seal plate and incubate at 37 °C for 30 minutes.
  5. Add 50 µL of Stop Solution to all wells and mix.  For Control well(s), add 25 µL of dilute Substrate after addition of Stop Solution.
  6. Seal plate and incubate at room temperature for 15 minutes.
  7. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
  8. 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 # A9891).

Per Well:

  • rhSIRT5: 0.025 µg
  • Substrate: 25 µM
  • beta -NAD: 0.5 mM

FAQs

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