Recombinant Mouse CD39L2/ENTPD6 Protein, CF

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

Recombinant Mouse CD39L2/ENTPD6 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the 5’-phosphate groups from the substrate guanosine-5’-diphosphate (GDP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996). The specific activity is >80,000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse CD39L2/ENTPD6 protein
  Lys33-Leu455, with an N-terminal 6-His tag
• Accession #: NP_742115
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Entpd6
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 47 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: 50-60 kDa, 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: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 5 mM CaCl₂, pH 7.5
  • Recombinant Mouse CD39L2/ENTPD6 (rmCD39L2) (Catalog # 6288-EN)
  • Substrate: GDP (Sigma, Catalog # G-7127), 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. Dilute rmCD39L2 to 0.01 µg/mL in Assay Buffer (note: for optimum activity minimize the number of dilution steps).
  2. 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).
  3. 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.
  4. Load 25 µL of 0.01 μg/mL rmCD39L2 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/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:
  • rmCD39L2: 0.00025 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 CD39L2/ENTPD6 Protein, CF

• CD39 antigen-like 2
• CD39L2
• CD39L2NTPDase-6
• CD39-like 2
• dJ738P15.3
• DKFZp781G2277
• DKFZp781K21102
• EC 3.6.1.6
• ectonucleoside triphosphate diphosphohydrolase 6 (putative function)
• ectonucleoside triphosphate diphosphohydrolase 6 (putative)
• ectonucleoside triphosphate diphosphohydrolase 6
• ENTPD6
• FLJ36711
• IL-6SAG
• IL6ST2
• interleukin 6 signal transducer-2
• NTPD3
• NTPDase 6

Background

CD39L2, also known as ectonucleoside triphosphate diphosphohydrolase-6 (ENTPD-6) and NTPDase-6, is a nucleoside phosphohydrolase of the CD39 family of enzymes that is present on the surface of cells (1). This Type II membrane protein also exists as a soluble, secreted protein. CD39L2 displays a preference for the nucleoside-5’-diphosphates GDP and IDP over CDP and UDP (2). Nucleoside-5’-triphosphates are also hydrolyzed, but at much lower rates. CD39L2 is expressed at its highest levels in cardiac muscle and in heart capillary endothelial cells (3). CD39L2 may play a functional role in the regulation and recruitment of platelet activation in the heart. The cytoplasmic and transmembrane domains of rmCD39L2 were replaced with a signal sequence, resulting in the secretion of the recombinant protein.

1. Chadwick, B.P. and A.M. Frischauf (1998) Genomics. 50:357.
2. Hicks-Berger, C.A. et al. (2000) J.Biol. Chem. 275:34041.
3. Yeung, G. et al. (2000) Biochemistry. 39:12916.

Bioinformatics

• Gene Symbol: Entpd6
• Uniprot:
  • NP_742115()