Recombinant Human CD73 (carrier-free)

Product Details

Source

Human CD73, amino acid (Trp27 - Lys547) (Accession: P21589) was expressed in CHO cells. The C-terminus contains 6His tag.

Molecular Mass

The 527 amino acid recombinant protein has a predicted molecular mass of approximately 58.6 kD. The DTT-reduced protein migrates at approximately 62 kD by SDS-PAGE. The predicted N-terminal amino acid is Trp.

Purity

> 95%, as determined by Coomassie stained SDS-PAGE

Formulation

0.22 µm filtered protein solution is in 0.3 M NaCl, 20 mM TRIS, pH 7.5

Endotoxin Level

Less than 0.1 EU per µg cytokine as determined by the LAL method

Concentration

25 µg size is bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.

Storage & Handling

Unopened vial can be stored at -20°C or -70°C for six months. For maximum results, quick spin vial prior to opening. Avoid repeated freeze/thaw cycles.

Activity

Recombinant human CD73 activity is 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. The specific activity is >30,000 pmol/min/μg, as measured in the presence of 0.001 ug of human CD73.

Application

Bioassay

Application Notes

Human CD73 Activity Assay
 

Recombinant human CD73 activity is 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. The specific activity is > 10,000 pmol/min/μg, as measured in the presence of 0.001 μg of human CD73.
 

Materials
 

Assay buffer: 25 mM Tris, 5 mM MgCl2, pH 7.5

Human 5’-Nucleotidase/CD73 

Substrate: Adenosine monophosphate (AMP) (Sigma, Cat. No. A1752), 5 mM stock in deionized water

Malachite Green Phosphate Detection Kit (Sigma, Cat. No. MAK307-1KT)

96-well Clear Plate (Costar, Cat. No. 92592)

Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Activity Assay Procedures
 

1. Prepare the dilution of human CD73 at 0.04 µg/mL in assay buffer.

2. In assay buffer, prepare a phosphate standard curve, first concentration should be 100 µM.  The standard curve should contain a range of 0.039 to 2.5 nmol per well. 

3. In a clear plate, load 25 µL of prepared human CD73 (0.04 µg/mL), standard curve, and blanks of assay buffer.

4. In assay buffer, dilute the substrate to 100 µM. Add 25 µl of substrate to all the wells.  Ensure to mix well. 

5. Parafilm the plate and incubate at 37ᵒC for 20 minutes. 

6. Make a 1:100 dilution of Malachite Green Solution B in Solution A, respectively. For example, 10 µL Solution B should be mixed with 1 mL of Solution A. Mix well. 

7. Add 10 µL of the mixture to each well. Mix well. At room temperature, incubate for 20 minutes. 

8. Read at wavelength of 620 nm (absorbance) in endpoint mode. 

 

Calculate the specific activity using the following formula:
 

Specific Activity (pmol/min/µg) =  (Phosphate released* (nmol) x (1000 pmol/1 nmol)) 

                                                               (Incubation time (min) X amount of enzyme (µg))

 

• Adjusted for the substrate blank and derived from the phosphate standard curve. 

Final Assay conditions per well:

hCD73: 0.001 ug 

Substrate: 35.7 µM

 

BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are validated in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com

Antigen Details

Structure

Monomer, ectonucleotidase

Distribution

Epithelial cells (respiratory tract), endothelial cells, smooth muscle cells, cardiac myocytes, and cancer cells. Lymphocytes, Tregs, anergic CD4+ T cells, effector T cells, macrophages, and dendritic cells.

Function

Regulates AMP and nicotinamide adenine dinucleotide (NAD+) metabolism. Hydrolyzes extracellular adenosine monophosphate into adenosine and inorganic phosphate. It is upregulated by hypoxia, TGFβ, IFNs, TNF, IL-1β, and prostaglandin E2.

Interaction

ECM proteins, fibronectin, tenascin C, and collagen

Ligand/Receptor

AMP

Bioactivity

Recombinant human CD73 activity is measured by its ability to hydrolyze the 5’-phosphate group from the substrate adenosine-5’-monophosphate (AMP).

Cell Type

B cells, Dendritic cells, Endothelial cells, Epithelial cells, Mesenchymal Stem Cells, T cells, Tregs

Biology Area

Costimulatory Molecules, Immunology, Stem Cells

Molecular Family

Adhesion Molecules, CD Molecules

Antigen References

1. Deaglio S, et al., 2007. J Exp Med. 204:1257.
2. Heuts DP, et al., 2012. Chembiochem 13:2384.
3. Pettengill M, et al., 2013. J Biol Chem. 288:27315.
4. Antonioli L, et al., 2016. Trends Cancer 2:95.
5. Allard B, et al., Immunol Rev. 276:121.
6. Kordaß T, et al., 2018. Front Immunol 9:813.
7. Zeiner J, et al., 2019. J Mol Med. 97:341. 

Gene ID

4907 View all products for this Gene ID

UniProt

View information about CD73 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Material Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Why choose BioLegend recombinant proteins?

     • Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
     • Greater than 95% Purity or higher, tested on every lot of product.
     • 100% Satisfaction Guarantee for quality performance, stability, and consistency.
     • Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
     • Bulk and customization available. Contact us.
     • Learn more about our Recombinant Proteins.

How does the activity of your recombinant proteins compare to competitors?

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

What is the specific activity or ED50 of my recombinant protein?

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

Have your recombinants been tested for stability?

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Does specific activity of a recombinant protein vary between lots?

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?

Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)

Certificate of Analysis