Recombinant Human 5'-Nucleotidase/CD73 Protein, CF

Catalog # Availability Size / Price Qty
5795-EN-010
5795-EN-500

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

Product Specifications

Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
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
Analysis
Trp27
Structure / Form
Dimer
Predicted Molecular Mass
59 kDa
SDS-PAGE
61-62 kDa, reducing conditions

Product Datasheets

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5795-EN

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

5795-EN

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, CaCl2 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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • 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
Reconstitution Calculator

Reconstitution Calculator

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Background: 5'-Nucleotidase/CD73

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.

References
    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.
    Entrez Gene IDs
    4907 (Human); 23959 (Mouse); 58813 (Rat); 102114976 (Cynomolgus Monkey)
    Alternate Names
    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

    Citations for Recombinant Human 5'-Nucleotidase/CD73 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.

    5 Citations: Showing 1 - 5
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    1. Ocular Hypotensive Properties and Biochemical Profile of QLS-101, a Novel ATP-Sensitive Potassium (KATP) Channel Opening Prodrug
      Authors: CL Pervan-Ste, U Roy Chowdh, HK Sookdeo, RA Casale, PI Dosa, TM Htoo, MP Fautsch, BM Wirostko
      Investigative Ophthalmology & Visual Science, 2022;63(4):26.
      Species: Human
      Sample Types: Small Molecule
      Applications: Bioassay
    2. A Novel CD73 Inhibitor SHR170008 Suppresses Adenosine in Tumor and Enhances Anti-Tumor Activity with PD-1 Blockade in a Mouse Model of Breast Cancer
      Authors: S Liu, D Li, J Liu, H Wang, I Horecny, R Shen, R Zhang, H Wu, Q Hu, P Zhao, F Zhang, Y Yan, J Feng, L Zhuang, J Li, L Zhang, W Tao
      OncoTargets and Therapy, 2021;14(0):4561-4574.
      Species: Human
      Sample Types: Recombinant Proteins
      Applications: Bioassay
    3. A Novel CD73 Inhibitor SHR170008 Suppresses Adenosine in Tumor and Enhances Anti-Tumor Activity with PD-1 Blockade in a Mouse Model of Breast Cancer
      Authors: S Liu, D Li, J Liu, H Wang, I Horecny, R Shen, R Zhang, H Wu, Q Hu, P Zhao, F Zhang, Y Yan, J Feng, L Zhuang, J Li, L Zhang, W Tao
      OncoTargets and Therapy, 2021;14(0):4561-4574.
      Species: Human
      Sample Types: Small Molecule
      Applications: Bioassay
    4. Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39
      Authors: SA Goueli, K Hsiao
      PLoS ONE, 2019;14(10):e0220094.
      Species: Human
      Sample Types: Recombinant Protein
      Applications: Bioassay
    5. Tiamulin inhibits breast cancer growth and pulmonary metastasis by decreasing the activity of CD73
      Authors: X Yang, S Pei, H Wang, Y Jin, F Yu, B Zhou, H Zhang, D Zhang, D Lin
      BMC Cancer, 2017;17(1):255.
      Species: Human
      Sample Types: Whole Cells
      Applications: Bioassay

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

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    Recombinant Human 5'-Nucleotidase/CD73 Protein, CF
    By Anonymous on 03/16/2020
    Application: In vitro bioactivity in cell culture