Recombinant Human CD39L2/ENTPD6 Protein, CF
Recombinant Human CD39L2/ENTPD6 Protein, CF Summary
Lys61-Ser484, with an N-terminal 6-His tag
Accession # O75354
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl, CaCl2 and Glycerol.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 25 mM Tris, 5 mM CaCl2, pH 7.5
- Recombinant Human CD39L2/ENTPD6 (rhCD39L2) (Catalog # 4399-EN)
- Substrate: GDP (Sigma, Catalog # G-7127), 100mM stock in deionized H2O
- 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 rhCD39L2 to 0.02 µg/mL in Assay Buffer.
- Prepare a standard curve from 1 M Phosphate Standard supplied in the Malachite Green Phosphate Detection Kit:
- Add 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock.
- Add 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 in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Load 25 µL of 0.02 μg/mL rhCD39L2 and the standard curve into a plate. Include a Substrate Blank containing Assay Buffer.
- Dilute the Substrate to 100 µM in Assay Buffer.
- Add 25 µL of the 100 µM Substrate to all wells and mix well.
- Cover the plate with parafilm or a plate sealer and incubate at 37 °C 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:
- rhCD39L2: 0.0005 µg
- Substrate: 35.7 µM
Ectonucleoside triphosphate diphosphohydrolase-6 (NTPDase-6) is a secreted nucleoside phosphohydrolase of the CD39 family of enzymes (1). hNTPDase-6 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. hNTPDase-6 has a broad tissue distribution, its mRNA has been detected in all human tissues tested (1).
- Chadwick, B.P. and A.M. Frischauf (1998) Genomics. 50:357.
- Hicks-Berger, C.A. et al. (2000) J. Biol. Chem. 275:34041.
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Phosphatase Activity Assays
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