Recombinant Human 5'-Nucleotidase/CD73 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human 5'-Nucleotidase/CD73 Protein, CF Summary

Details of Functionality
Measured by its ability to hydrolyze the 5’-phosphate group from the substrate adenosine-5’-monophosphate (AMP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996). The specific activity is >15,000 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human 5'-Nucleotidase/CD73 protein
Trp27-Lys547, with a C-terminal 6-His tag, Trp27-Lys547
Accession #
N-terminal Sequence
Trp27
Structure / Form
Dimer
Protein/Peptide Type
Recombinant Enzymes
Gene
NT5E
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
59 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
61-62 kDa, reducing conditions
Publications
Read Publications using
5795-EN in the following applications:

Packaging, Storage & Formulations

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.
Buffer
Supplied as a 0.2 μm filtered solution in Tris, NaCl, CaCl2 and Glycerol.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Assay Procedure
  • Assay Buffer: 25 mM Tris, 5 mM MgCl2, pH 7.5
  • Recombinant Human 5’-Nucleotidase/CD73 (rhCD73) (Catalog # 5795-EN)
  • Substrate: Adenosine monophosphate (AMP) (Sigma, Catalog # A1752), 5 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
  1. Dilute rhCD73 to 0.04 µg/mL in Assay Buffer.
  2. Prepare a standard curve from the 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer 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 (this is the first dilution to use as a standard).
  3. Perform six additional one-half serial dilutions of the 100 µM phosphate stock. The standard curve has a range of 0.039 to
    2.5 nmol per well.
  4. Load 25 µL of 0.04 µg/mL rhCD73, standard curve, and blanks (Assay Buffer) into a plate.
    Dilute Substrate to 100 µM in Assay Buffer.
  5. Add 25 µL of the Substrate to all wells. Mix well.
  6. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 20 minutes.
  7. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  8. Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  9. Read plate at 620 nm (absorbance) in endpoint mode.
  10. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Phosphate released* (nmol) x (1000 pmol/1 nmol)
Incubation time (min) x amount of enzyme (µg)

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank.

Per Well:
  • rhCD73: 0.001 µg
  • Substrate: 35.7 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human 5'-Nucleotidase/CD73 Protein, CF

  • 5' nucleotidase (CD73)
  • 5'-NT
  • 5-NT
  • 5'-Nucleotidase
  • 5'-nucleotidase, ecto (CD73)
  • CD73 antigen
  • CD73
  • E5NT
  • EC 3.1.3.5
  • ecto-5'-nucleotidase
  • eN
  • eNT
  • NT
  • NT55'-nucleotidase
  • NT5E
  • NTE
  • Purine 5-Prime-Nucleotidase

Background

CD73, an ecto-5'-Nucleotidase, is an ectoenzyme expressed by most cell types (1). The 5'-Nucleotidase activity of CD73 converts extracellular nucleoside‑5'‑monophosphates to nucleosides, with AMP as the preferred substrate. CD73 is one of several enzymes responsible for the production of extracellular adenosine, a signaling molecule that is involved in responses to inflammation and tissue injury (2). CD73 is a lymphocyte maturation marker that has functions independent of its catalytic activity. These functions include the adhesion of B-cells to follicular dendritic cells and T-cell signaling (3, 4). CD73 is also a regulator of leukocyte extravasation, a function that requires its 5'-Nucleotidase activity (5). The native enzyme is a homodimer bound to the cell membrane through a glycosyl phophatidylinositol (GPI) anchor.
    1. Resta, R. et al. (1998) Immunol. Rev. 161:95.
    2. Pilcher, M. et al. (2003) J. Biol. Chem. 278:13468.
    3. Airas, L. (1998) Leuk. Lymphoma 29:37.
    4. Resta, R. and Thompson. L.F. (1997) Cell Signal. 9:131.
    5. Jalkanen, S. and Salmi, M. (2008) Arterioscler. Thromb. Vasc. Biol. 28:18.

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    Publications for 5'-Nucleotidase/CD73 (5795-EN)(2)

    We have publications tested in 1 confirmed species: Human.

    We have publications tested in 1 application: Bioassay.


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    (2)
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    FAQs for 5'-Nucleotidase/CD73 (5795-EN). (Showing 1 - 1 of 1 FAQs).

    1. We are interested in CD73 antibodies that could react with dog, please let me know which products in your catalog you would recommend.
      • We do not currently carry any CD73 antibodies that have been validated for use in dog samples. However, if you are interested in testing any of our CD73 antibodies with dog samples you would be eligible for our Innovators Reward Program, in which you can get a discount voucher for the purchase price of the product. Please contact us at innovators@novusbio.com with any questions regarding this program.

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    Bioinformatics

    Gene Symbol NT5E
    Uniprot