Recombinant Mouse CD39L3/ENTPD3 Protein, CF Summary
Gln44-Pro485, with a C-terminal 6-His tag
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 Mouse CD39L3/ENTPD3 (rmCD39L3) (Catalog # 4464-EN)
- Substrate: Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 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
- Dilute rmCD39L3 to 0.02 µg/mL in Assay Buffer.
- 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.)
- 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 rmCD39L3 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 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/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:
- rmCD39L3: 0.0005 µg
- Substrate: 35.7 µM
Ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase-3) is an integral membrane protein with an extracellular catalytic domain (1). rhNTPDase-3 was expressed as a protein lacking its N- and C-terminal transmembrane domains, resulting in the secretion of the soluble rhNTPDase-3 ectodomain. NTPDase-3 hydrolyzes the b- and g phosphate groups of nucleotides, preferring ATP, ADP, UTP, and UDP as substrates (1). Through its hydrolysis of extracellular nuceotides, NTPDase-3 is important for the regulation of purinergic signaling (2). The enzyme is expressed at its highest levels in brain, pancreas, spleen, and prostate tissues (3). In the brain, NTPDase-3 may play a role in the regulation of feeding, sleep, and other behaviors (4).
- Lavoie, E.G. et al. (2004) Biochem. Pharmacol. 67:1917.
Crawford, P.A. et al. (2007) Arch. Biochem. Biophys. 457:7.
- Chadwick, B.P. and A.M. Frischauf (1998) Genomics 50:357.
- Belcher, S.M. et al. (2006) Neuroscience 137:1331.
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Phosphatase Activity Assays
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