Recombinant Mouse CD39L1/ENTPD2 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse CD39L1/ENTPD2 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the 5’-phosphate groups from the substrate adenosine-5’-triphosphate (ATP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996). The specific activity is >9,000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse CD39L1/ENTPD2 protein
  Cys26-Ser462, with a C-terminal 6-His tag
• Accession #: O55026
• N-terminal Sequence: Cys26
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Entpd2
• Purity: >95%, 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

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 49 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: 60-80 kDa, reducing conditions

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: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 5 mM CaCl₂, pH 7.5
  • Recombinant Mouse CD39L1/ENTPD2 (rmCD39L1) (Catalog # 5797-EN)
  • Substrate: Adenosine triphosphate (ATP) (Sigma, Catalog # A-7699), 10 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. Prepare a standard curve from 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 in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
  3. Dilute rmCD39L1 to 0.08 µg/mL in Assay Buffer.
  4. Load 25 µL of 0.08 µg/mL rmCD39L1 and the standard curve into a plate. Include a Substrate Blank containing Assay Buffer.
  5. Dilute the Substrate to 100 µM in Assay Buffer.
  6. Add 25 µL of the 100 µM Substrate to all wells and mix well.
  7. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
  8. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  9. Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  10. Read plate at 620 nm (absorbance) in endpoint mode.
  11. 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:
  • rmCD39L1: 0.002 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 Mouse CD39L1/ENTPD2 Protein, CF

• CD39 antigen-like 1
• CD39L1
• CD39L1NTPDase 2
• CD39-like-1
• EC 3.6.1
• EC 3.6.1.-
• EC 3.6.1.5
• Ecto-ATP diphosphohydrolase 2
• Ecto-ATPase 2
• Ecto-ATPase
• Ecto-ATPDase 2
• ectonucleoside triphosphate diphosphohydrolase 2
• ENTPD2
• NTPD2
• NTPDase 2
• NTPDase-2

Background

CD39L1, also known as ENTPD2 and NTPDase2, is an ecto-nucleotidase belonging to the CD39 family. It is found on the surface of vascular adventitial cells and accessory vascular cells (1). CD39L1 is a Ca²⁺- and Mg²⁺-dependent enzyme that activates platelets by preferentially converting ATP to ADP (2). CD39L1 plays a role in regulating thrombosis and inflammation (3). It is considered to be a therapeutic target for thromboregulation and the treatment of vascular inflammation (2, 4).

1. Zimmermann, H. et al. 2000 Proceedings of the Second International Workshop on Ecto-ATPases and Related Ectonucleotidases:18.        
2. Robson, S.C. et al. 2001 Drug Dev. Res. 53:193.  
3. Marcus, A.J. et al. 2005 Semin. Thromb. Hemost. 31:234.  
4. Sevigny, J. et al. 2002 Blood 99:2801.

Bioinformatics

• Gene Symbol: Entpd2
• Uniprot:
  • O55026()