Recombinant Human NMNAT-1 Protein, CF Summary
Product Specifications
Met1-Thr279, 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.
5865-NT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol 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, pH 7.5
- Recombinant Human NMNAT-1 (rhNMNAT-1) (Catalog # 5865-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-1 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 Assay Buffer.
- In a plate, load 50 µL of 0.5 ng/µL rhNMNAT-1, 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-1: 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-1
NMNAT-1 is expressed in the nuclei of all human tissues, with highest expression in skeletal muscle, heart, kidney, pancreas, and brain (1). The enzyme transfers adenylate from ATP to nicotinamide ribonucleotide or nicotinate ribonucleotide to generate NAD+ or deamido-NAD+, and is an essential enzyme for the production of nuclear NAD+ (2). Nuclear NAD+ is required by poly(ADP-ribose) polymerase 1 (PARP-1), which poly-ADP-ribosylates chromatin in response to DNA strand breaks. NMNAT-1 is known to interact with PARP-1, resulting in its activation, but this interaction with PARP-1 is prevented when NMNAT-1 is phosphorylated at Ser136 (3). Nuclear NAD+ levels are also important for the regulation of SIR2 histone deacetylases (4). A naturally occurring Ube4b/NMNAT-1 chimeric protein is directly involved in slowing the degeneration of injured neurons in mice (5). NMNAT activity is required for the activation of tiazofurin, a drug used to treat leukemia (6). Two other NMNAT enzymes are present in humans. NMNAT-2 is localized in the Golgi complex and cytoplasm, and NMNAT-3 is a mitochondrial enzyme (7).
- Emanuelli, M. et al. (2001) J. Biol.Chem. 276:406.
- Schweiger, M. et al. (2001) FEBS Lett. 492:95.
- Berger, F. et al. (2007) Proc. Natl. Acad. Sci. USA 104:3765.
- Revollo, J.R. et al. (2004) J. Biol. Chem. 279:50754.
- Mack, T.G. et al. (2001) Nature Neurosci. 4:1199.
- Boulton, S. et al. (1997) Br. J. Cancer 76:845.
- Berger, F. et al. (2005) J. Biol. Chem. 280:36334.
Citation for Recombinant Human NMNAT-1 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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Expression and regulation of nampt in human islets.
Authors: Kover K, Tong P, Watkins D, Clements M, Stehno-Bittel L, Novikova L, Bittel D, Kibiryeva N, Stuhlsatz J, Yan Y, Ye S, Moore W
PLoS ONE, 2013-03-11;8(3):e58767.
Species: Human
Sample Types: Cell Culture Supernates
Applications: Enzyme Assay
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