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

Additional Information
Fc Chimera
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 >12,500 pmol/min/μg, as measured under the described conditions.
Source
Human embryonic kidney cell, HEK293-derived human 5'-Nucleotidase/CD73 protein
Human CD73
(Trp27-Ser549)
Accession # AAH65937.1
GGIEGRMDMouse IgG2a
(Pro100-Lys330)
N-terminusC-terminus
Accession #
N-terminal Sequence
Trp27
Protein/Peptide Type
Recombinant Enzymes
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Note
<0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Dilutions
  • Enzyme Activity
Theoretical MW
85 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
84-93 kDa under reducing conditions.

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -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 Fc Chimera (rhCD73/mFc) (Catalog # 11415-EN)
  • Substrate: Adenosine monophosphate (AMP), 5 mM stock in deionized water
  • Malachite Green Phosphate Detection Kit  (Catalog # DY996)
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader with Absorbance Read Capability
  1. 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).
  2. Perform six additional one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Dilute rhCD73/mFc to 0.16 µg/mL in Assay Buffer.
  4. Dilute AMP to 200 µM in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of 0.16 µg/mL rhCD73/mFc into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  7. Start the reactions by adding 25 µL of 200 µM AMP to all wells, excluding the standard curve and curve blank.  
  8. Seal plate and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells used, including standard curve. Mix briefly.
  10. Add 100 µL of deionized water to all wells used, including standard curve. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells used, including standard curve. Mix briefly.
  12. Seal plate and incubate at room temperature for 20 minutes.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. 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 Reaction
  • rhCD73/mFc:  0.004 µg 
  • Substrate:  100 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 Fc 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, known as ecto-5'-Nucleotidase, converts extracellular nucleoside 5' monophosphates to nucleosides, with AMP as its preferred substrate (1). CD73 is a zinc-dependent, 70 kDa homodimeric enzyme bound to the cell membrane through a glycosyl phophatidylinositol (GPI) anchor. It is composed of an N-terminal domain containing metal binding sites linked via small hinge region to a C-terminal domain containing the substrate binding site and dimerization interface (2). It is expressed by most cell types (3) and is widely expressed in tumor cell lines as well as upregulated in cancerous tissues (4, 5). CD73 is a key enzyme responsible for a rate-limiting step in the generation of extracellular adenosine. Adenosine is a molecule that signals through activation of purinergic receptors and results in an immunosuppressive role in the tumor microenvironment (4, 6). CD73 has been implicated in many pathological processes including immunomodulation and inflammation (7, 8) tumor growth and metastasis (9-13) making CD73 a potential drug target in cancer. Targeting CD73 inhibition has resulted in numerous reports of favorable antitumor effects (4, 5, 12). Consequently, therapeutic approaches have been tested using knockdown, gene silencing and anti-CD73 therapies (11, 14) as well as small molecule inhibitors (14, 15).
  1. Zimmermann, H. et. al. (2012) Purinergic Signal. 8:437.
  2. Knapp, K. et. al. (2012) Structure. 20:2161.
  3. Resta, R. et. al. (1998) Immunol. Rev. 161:95. 
  4. Jin, D. et. al. (2010) Cancer Res. 70:2245.
  5. Zhang, B. (2010) Cancer Res. 70:6407.
  6. Picher, M. et. al. (2003) J. Biol. Chem. 278:13468. 
  7. Antonioli, L. et. al. (2012) Curr. Drug Targets 13:842.
  8. Eltzchig, H. K. et. al. (2012) N. Engl. J. Med. 367:2322.
  9. Bavaresco, L. et. al. (2008) Mol. Cell Biochem. 319:61.
  10. Yegutkin, G. G. et. al. (2011) Eur. J. Immunol. 41:1231.
  11. Stagg, J. et. al. (2012) Cancer Res. 72:2190.
  12. Ghalamfarsa, G. et. al. (2019) Expert Opin. Ther. Targets. 23:127.
  13. Gao, Z. W. et. al. (2014) Biomed. Res. Int. 2014:460654.
  14. Young, A. et. al. (2014) Cancer Discov. 4:879.
  15. McManus, J. et. al. (2018) SLAS Discov. 23:264.

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FAQs for 5'-Nucleotidase/CD73 (11415-EN). (Showing 1 - 1 of 1 FAQ).

  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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