Recombinant Mouse ENPP-7/Alk-SMase Protein, CF Summary
Ala22-Gln421, with a C-terminal 10-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 and NaCl.|
|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: 50 mM Tris, 10 mM Sodium Taurocholate (Sigma, Catalog # T4009), 150 mM NaCl, pH 9.0
- rhTNAP Buffer: 50 mM Tris, 2 mM MgCl2, pH 9.0
- Recombinant Mouse ENPP‑7/Alk‑SMase (rmENPP-7) (Catalog # 5160-EN)
- Substrate: Sphingomyelin (egg, chicken) (SPM) (Avanti Polar Lipids, Inc., Catalog # 860061C), 35 mM in chloroform
- Phosphocholine Chloride (Sigma, Catalog # P0378), 100 mM stock in deionized water
- Recombinant Human Alkaline Phosphatase/ALPL/TNAP (rhTNAP) (Catalog # 2909-AP)
- 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 SPM to 0.4 mM in Assay Buffer. Note: Heat buffer to 100 °C and then add SPM to help keep SPM solubilized. Vortex well.
- Dilute rmENPP-7 to 0.1 µg/mL in Assay Buffer.
- Mix equal volumes of diluted SPM and diluted rmENPP-7.
- Incubate at 37 °C for 30 minutes. As a Substrate Control substitute SPM with Assay Buffer and keep on ice during incubation.
- Stop the reaction by heating all vials at 95-100 °C for 6 min. Cool on ice for 1 min followed by brief centrifugation in microcentrifuge.
- Prepare a standard curve from the 100 mM Phosphocholine Chloride stock by adding 10 µL of the 100 mM Phosphocholine Chloride Standard to 990 µL of Assay Buffer for a 1 mM stock. Continue by adding 100 µL of the 1 mM Phosphocholine Chloride stock to 900 µL of Assay Buffer for a 100 µM stock (This is the first dilution to use as a standard).
- Make the following serial dilutions from the 100 µM Phosphocholine Chloride stock in Assay Buffer to complete standard curve: 80, 60, 40, 20, 10 and 5 μM.
- Dilute rhTNAP to 4 µg/mL in rhTNAP Buffer.
- Combine 100 µL of the 4 µg/mL rhTNAP with 100 µL each of the diluted rmENPP-7/SPM mixture, the dilutions of the standard curve, and the Substrate Control.
- Incubate at room temperature for 20 min.
- Mix equal volumes of the Malachite Green Reagents A and B together for enough volume to add 80 µL of the mixture to all vials.
- Add the Malachite Green Reagent A and B mixture to vials and vortex.
- Incubate at room temperature for 10 min. Note: Do not let mixtures incubate longer, precipitation may occur.
- Load 70 µL from each vial into a plate.
- 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 the Substrate Control.Per Well:
- rmENPP-7: 0.00125 µg
- Sphingomyelin: 0.071 mM
- Phosphocholine Chloride Curve: 0, 125, 250, 500, 1000, 1500, 2000, and 2500 pmol
ENPP-7 (ectonucleotide pyrophosphatase/phosphodiesterase 7), also known as alkaline sphingomyelinase (Alk-SMase) is expressed in the intestines and in bile (1). It shares 30 - 36% homology with the members of the nucleotide pyrophosphatase/phosphodiesterese (NPP) family while sharing no similarities with neutral or acid SMase (2). Its main function is the digestion of dietary sphingomyelin by hydrolyzing sphingomyelin into ceramide and phosphorylcholine. ENPP-7 is reported to hydrolyse and inactivate platelet-activating factor (PAF) by a phospholipase C-type activity (3). Studies show a decrease in ENPP-7 activity in human colorectal adenocarcinomas and human colorectal carcinomas, which indicate a potential role of ENPP-7 in human colon cancer (4, 5).
- Duan, R-D. et al. (2006) Biochim. Biophys. 1761:281.
- Duan, R-D. et al. (2003) J. Biol. Chem. 278:38528.
- Wu, J. et al. (2006) Biochem J. 394:299.
- Hertervig, E. et al. (1996) Cancer. 79:448.
- Hertervig, E. et al. (1999) Br. J. Cancer. 81:232.
Citation for Recombinant Mouse ENPP-7/Alk-SMase Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
Non-invasive imaging of tumors by monitoring autotaxin activity using an enzyme-activated near-infrared fluorogenic substrate.
Authors: Madan, Damian, Ferguson, Colin G, Lee, Won Yong, Prestwich, Glenn D, Testa, Charles
PLoS ONE, 2013;8(11):e79065.
Sample Types: Complex Sample Type
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Phosphatase Activity Assays
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