Recombinant Human NMNAT-2 Protein, CF

R&D Systems | Catalog # 6279-NT

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Key Product Details

  • R&D Systems Sf 21 (baculovirus)-derived Recombinant Human NMNAT-2 Protein (6279-NT)
  • 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

Q9BZQ4

Applications

Enzyme Activity
View all NMNAT-2 Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human NMNAT-2 protein
Met1-Glu307, with a C-terminal 6-His tag

Purity

>70%, 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.

N-terminal Sequence Analysis

Thr2, Thr133, Thr145

Predicted Molecular Mass

35 kDa

SDS-PAGE

34-38 kDa, reducing conditions

Activity

Measured by the production of NAD+, which is converted to NADH by alcohol dehydrogenase.
The specific activity is >1,500 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

6279-NT
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij-35 and DTT.
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.

Background: NMNAT-2

Humans produce three nicotinamide mononucleotide adenylyltransferase (NMNAT) enzymes. All three enzymes transfer adenylate from ATP to nicotinamide ribonucleotide or nicotinate ribonucleotide to generate NAD+ or deamido-NAD+, and are important enzymes in the NAD biosynthetic pathway (1). The three enzymes differ in tissue expression patterns and subcellular location, indicating that they each play a unique role in NAD homeostasis (2). NMNAT-2 is expressed primarily in the central nervous system and in muscle tissue in contrast to NMNAT-1, which has a broad tissue distribution (3). NMNAT-2 is found in the cytosol and Golgi complex, while NMNAT-1 is a nuclear enzyme and NMNAT-3 is mitochondrial (1). A number of studies have implicated the NMNAT proteins in cancer and neurodegenerative diseases (4). NMNAT‑2 has been shown to be essential for the maintenance of healthy axons. When the level of this labile protein falls below a critical threshold in axons, the process of axonal degeneration begins (5). The axon-protective function of NMNAT-2 is dependent on its NAD+ synthesis activity (6).

 

References

  1. Berger, F. et al. (2005) J. Biol. Chem. 280:36334.
  2. Raffaelli, N. et al. (2002) Biochem. Biophys. Res. Commun. 297:835.
  3. Yalowitz, J.A. et al. (2004) Biochem. J. 377:317.
  4. Lau, C. et al. (2009) Front. Biosci. 14:410.
  5. Gilley, J. and M.P. Coleman (2010) PLoS Biol. 8:1.
  6. Yan, T. et al. (2010) Neurochem. Int. 56:101.  

Long Name

Nicotinamide mononucleotide adenylyltransferase-2

Alternate Names

NMNAT2, PNAT2

Entrez Gene IDs

23057 (Human)

Gene Symbol

NMNAT2

UniProt

Q9BZQ4

Additional NMNAT-2 Products

Product Documents for Recombinant Human NMNAT-2 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human NMNAT-2 Protein, CF

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

For research use only

Citations for Recombinant Human NMNAT-2 Protein, CF

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Protocols

View specific protocols for Recombinant Human NMNAT-2 Protein, CF (6279-NT):

Materials
  • Assay Buffer: 50 mM HEPES, 5 mM DTT, pH 7.5
  • Recombinant Human NMNAT‑2 (rhNMNAT-2) (Catalog # 6279-NT)
  • beta -Nicotinamide mononucleotide ( beta -NMN) (Sigma, Catalog # N3501), 50 mM stock in deionized water
  • Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
  • Yeast Alcohol Dehydrogenase (ADH) (Sigma, Catalog # A3263), 5 mg/mL stock in 25 mM MES, 20% Glycerol, pH 6.5
  • 1 M Magnesium Chloride
  • 95-100% Ethanol
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhNMNAT-2 to 0.5 ng/µL in Assay Buffer.
  2. Prepare Substrate Mixture: 30 mM MgCl2, 1 mM beta -NMN, 4 mM ATP, 0.1 mg/mL ADH, and 2% Ethanol in 50 mM HEPES, pH 7.5.
  3. In a plate, load 50 µL of 0.5 ng/µL rhNMNAT-2, and start the reaction by adding 50 µL of Substrate Mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
  4. Read absorbance at a wavelength of 339 nm (bottom read) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

     *Adjusted for Substrate Blank 
     **Using the extinction coefficient 6220 M-1cm-1 
     ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD Per Well:
  • rhNMNAT-2: 0.025 µg
  • Substrate Mixture: 0.5 mM beta -NMN, 2 mM ATP, 15 mM MgCl2, 0.05 mg/mL ADH, and 1% Ethanol

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