Recombinant Human 5'-Nucleotidase/CD73 Protein, CF
Recombinant Human 5'-Nucleotidase/CD73 Protein, CF Summary
Product Specifications
Trp27-Lys547, with a C-terminal 6-His tag, Trp27-Lys547
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.
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.
|
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
- Dilute rhCD73 to 0.04 µg/mL in Assay Buffer.
- 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).
- 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. - 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. - Add 25 µL of the Substrate to all wells. Mix well.
- Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 20 minutes.
- Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 10 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/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
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.
- Resta, R. et al. (1998) Immunol. Rev.
161:95.
- Pilcher, M. et al. (2003) J.
Biol. Chem. 278:13468.
- Airas, L. (1998) Leuk. Lymphoma 29:37.
- Resta, R. and Thompson. L.F. (1997) Cell
Signal. 9:131.
- Jalkanen, S. and Salmi, M. (2008)
Arterioscler. Thromb. Vasc. Biol. 28:18.
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.
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Citations: Showing 1 - 6
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Assessment of ATP Metabolism to Adenosine by Ecto-Nucleotidases Carried by Tumor-Derived Small Extracellular Vesicles
Authors: Hong, CS;Menshikova, EV;Whiteside, TL;Jackson, EK;
Research square
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
CD73 inhibits cGAS-STING and cooperates with CD39 to promote pancreatic cancer
Authors: C Jacoberger, I Cousineau, Y Bareche, D Allard, P Chrobak, B Allard, S Pommey, N Messaoudi, Y McNicoll, G Soucy, S Koseoglu, R Masia, AC Lake, H Seo, CB Eeles, N Rohatgi, SC Robson, S Turcotte, B Haibe-Kain, J Stagg
Cancer Immunology Research, 2023-01-03;0(0):.
Species: N/A
Sample Types: Buffer
Applications: ELISA Standard -
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, 2022-04-01;63(4):26.
Species: Human
Sample Types: Small Molecule
Applications: Bioassay -
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, 2021-08-24;14(0):4561-4574.
Species: Human
Sample Types: Recombinant Proteins, Small Molecule
Applications: Bioassay -
Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39
Authors: SA Goueli, K Hsiao
PLoS ONE, 2019-10-25;14(10):e0220094.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay -
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, 2017-04-11;17(1):255.
Species: Human, Mouse
Sample Types: Whole Cells
Applications: Bioassay
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