Recombinant Human NMNAT-2 Protein, CF Summary
Product Specifications
Met1-Glu307, with a C-terminal 6-His tag
Analysis
Product Datasheets
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.
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.
|
Assay Procedure
- 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
- Dilute rhNMNAT-2 to 0.5 ng/µL in Assay Buffer.
- 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.
- 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.
- Read absorbance at a wavelength of 339 nm (bottom read) in kinetic mode for 5 minutes.
- 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
Reconstitution Calculator
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).
- Berger, F. et al. (2005) J. Biol. Chem. 280:36334.
- Raffaelli, N. et al. (2002) Biochem. Biophys. Res. Commun. 297:835.
- Yalowitz, J.A. et al. (2004) Biochem. J. 377:317.
- Lau, C. et al. (2009) Front. Biosci. 14:410.
- Gilley, J. and M.P. Coleman (2010) PLoS Biol. 8:1.
- Yan, T. et al. (2010) Neurochem. Int. 56:101.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.Citation for Recombinant Human NMNAT-2 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.
1 Citation: Showing 1 - 1
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Screening with an NMNAT2-MSD platform identifies small molecules that modulate NMNAT2 levels in cortical neurons
Authors: YO Ali, G Bradley, HC Lu
Sci Rep, 2017-03-07;7(0):43846.
Species: Mouse
Sample Types: Tissue Homogenates
Applications: ELISA (Standard)
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