Recombinant Mouse 5'-Nucleotidase/CD73 Protein, CF Summary
Trp29-Lys549, 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, 0.5 mM MgCl2, pH 7.5
- Recombinant Mouse 5'‑Nucleotidase/CD73 (rmCD73) (Catalog # 4488-EN)
- Substrate: AMP (Sigma, Catalog # A1752), 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 rmCD73 to 0.04 µg/mL in Assay Buffer.
- Prepare a standard curve from the 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. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Load 25 µL of 0.04 µg/mL rmCD73, standard curve, and blanks (Assay Buffer) into a plate.
Dilute Substrate to 200 µM in Assay Buffer.
- Add 25 µL of the Substrate to all wells. 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/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 Reaction:
- rmCD73: 0.001 µg
- Substrate: 100 µM
CD73, an ecto-5’-Nucleotidase, is an ectoenzyme that is attached to the cell membrane by a glycosyl phophatidylinositol anchor (1, 2). The enzyme is expressed by most cell types. The 5’-Nucleotidase activity of CD73 converts extracellular nucleoside-5’-monophosphates to nucleosides. CD73 is one of several enzymes responsible for the production of extracellular adenosine, a signaling molecule that is involved in responses to inflammation and tissue injury (3).
- Resta, R. et al. (1993) Gene 133:171.
- Resta, R. et al. (1998) Immunol. Rev. 161:95.
- Pilcher, M. et al. (2003) J. Biol. Chem. 278:13468.
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Phosphatase Activity Assays
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