Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, CF
Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, CF Summary
Product Specifications
Glu2-Phe219, with an N-terminal Met and 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.
6414-NH
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, DTT and Glycerol. |
| 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: 25 mM Tris, 5 mM MgCl2, pH 7.5
- Recombinant Human ADP‑Sugar Pyrophosphatase/NUDT5 (rhNUDT5) (Catalog # 6414-NH)
- Coupling Enzyme: 5'-Nucleotidase/CD73 (rhCD73) (Catalog # 5795-EN)
- Substrate: Adenosine 5’-diphosphate ribose (ADPR) (Sigma, Catalog # A0752), 20 mM stock in deionized water
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute Substrate to 2.5 mM in Assay Buffer.
- Dilute rhCD73 to 3.33 µg/mL in Assay Buffer.
- Dilute rhNUDT5 to 0.667 µg/mL in Assay Buffer.
- Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock.
- Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 15 µL of 0.667 µg/mL rhNUDT5 into the plate. Include a Control containing 15 µL of Assay Buffer.
- Add 15 µL of 3.33 µg/mL rhCD73 to the wells, excluding the standard curve and the curve blank.
- Start the reaction by adding 20 µL of 2.5 mM Substrate to the wells, exluding the standard curve and the curve blank.
- Cover the plate with parafilm or a plate sealer and incubate at room temperature for 30 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells.
- Add 100 µL of deionized water to all wells.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Phosphate released* (nmol) x (1000 pmol/nmol) |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
Per Well:- rhNUDT5: 0.01 µg
- rhCD73: 0.05 µg
- Substrate: 0.238 mM
Reconstitution Calculator
Background: ADP-Sugar Pyrophosphatase/NUDT5
ADP-Sugar Pyrophosphatase (NUDT5) is a member of the nudix superfamily of enzymes. Members of this family are pyrophosphohydrolases that act upon substrates with the general structure of a nucleoside (Nu) diphosphate (di) linked to another moiety, X (NDP-X) to yield NMP plus P-X (1). Human NUDT5 is a homodimeric enzyme present in the cytosol of most cell types (2). Glu166 and three magnesium ions are important for stabilizing the transition state during the hydrolysis of ADPR (3). NUDT5 has been suggested to play a role in regulating the intracellular levels of ADPR by NO activation through ADP-ribosylation at cysteine residues of the enzyme in macrophages (4). It also may play defensive role against the mutagenesis induced by oxidized deoxyribonucleosides (5, 6).
- McLennan, A.G. (2006) Cell Mol. Life Sci. 63:123.
- Zha, M. et al. (2006) J. Mol. Biol. 364:1021.
- Zha, M. et al. (2008) J. Mol. Biol. 379:568.
- Yu, H.N. et al. (2007) Biochem. Biophys. Res. Comm. 354:764.
- Hori, M. et al. (2010) Free Radic. Biol. Med. 48:1197.
- Kamiya, H. et al. (2009) DNA Repair (Amst) 8:1250.
Citation for Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, 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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Deletion of Annexin A1 in Mice Upregulates the Expression of Its Receptor, Fpr2/3, and Reactivity to the AnxA1 Mimetic Peptide in Platelets
Authors: O Zharkova, MF Salamah, MV Babak, E Rajan, LHK Lim, F Andrade, CD Gil, SM Oliani, LA Moraes, S Vaiyapuri
International Journal of Molecular Sciences, 2023-02-08;24(4):.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay
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