Recombinant Human 5'‑Nucleotidase/CD73 Fc Chimera
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Recombinant Human 5'-Nucleotidase/CD73 Fc Chimera Catalog Number: 11415-ENDESCRIPTION Source Human embryonic kidney cell, HEK293-derived human 5'-Nucleotidase/CD73 protein N-terminal Sequence Trp27 Analysis Predicted Molecular 85 kDa Mass SPECIFICATIONS SDS-PAGE 84-93 kDa under reducing conditions. Activity Measured by its ability to hydrolyze the 5'-phosphate group from the substrate adenosine-5'-monophosphate (AMP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996). The specific activity is >12,500 pmol/min/gg, as measured under the described conditions. Endotoxin Level <0.10 EU per 1 gg of the protein by the LAL method. Purity >90%, by SDS-PAGE under reducing conditions and visualized by silver stain. Formulation Supplied as a 0.2 gm filtered solution in Tris, NaCl, CaCl2 and Glycerol. See Certificate of Analysis for details. Activity Assay Protocol Materials • Assay Buffer: 25 mM Tris, 5 mM MgCl2, pH 7.5 • Recombinant Human 5-Nucleotidase/CD73 Fc Chimera (rhCD73/mFc) (Catalog # 11415-EN) • Substrate: Adenosine monophosphate (AMP), 5 mM stock in deionized water • Malachite Green Phosphate Detection Kit (Catalog # DY996) • 96-well Clear Plate (Catalog # DY990) • Plate Reader with Absorbance Read Capability Assay 1. Prepare a standard curve from the 1 M Phosphate Standard by adding 10 |jL of the 1 M Phosphate Standard to 990 |jL of Assay Buffer for a 10 mM stock. Continue by adding 10 jL of the 10 mM Phosphate stock to 990 jL of Assay Buffer for a 100 jM stock (this is the first dilution to use as a standard). 2. Perform six additional one-half serial dilutions of the 100 jM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well. 3. Dilute rhCD73/mFc to 0.16 jg/mL in Assay Buffer. 4. Dilute AMP to 200 jM in Assay Buffer. 5. Load 50 jL of each dilution of the standard curve into a plate. Include a curve blank containing 50 gL of Assay Buffer. 6. Load 25 jL of 0.16 jg/mL rhCD73/mFc into empty wells of the same plate as the curve. Include a Control containing 25 gL of Assay Buffer. 7. Start the reactions by adding 25 jL of 200 jM AMP to all wells, excluding the standard curve and curve blank. 8. Seal plate and incubate at 37 °C for 20 minutes. 9. Add 30 jL of the Malachite Green Reagent A to all wells used, including standard curve. Mix briefly. 10. Add 100 jL of deionized water to all wells used, including standard curve. Mix briefly. 11. Add 30 jL of the Malachite Green Reagent B to all wells used, including standard curve. Mix briefly. 12. Seal plate and incubate at room temperature for 20 minutes. 13. Read plate at 620 nm (absorbance) in endpoint mode. 14. Calculate specific activity: Specific Activity (pmol/min/jg) = Phosphate released* (nmol) x (1000 pmol/nmol) Incubation time (min) x amount of enzyme (jg) *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control. Final Assay Per Reaction PREPARATION AND STORAGE 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. • 6 months from date of receipt, -20 to -70 °C as supplied. • 3 months, -20 to -70 °C under sterile conditions after opening. biotechne Global bio-techne.com info@bio-techne.com techsupport@bio-techne.com TEL +1612 379 2956 USA TEL 800 343 7475 Canada TEL 855 668 8722 China TEL +86 (21) 52380373 Europe | Middle East | Africa TEL +44 (0)1235 529449

Recombinant Human 5'-Nucleotidase/CD73 Fc Chimera Catalog Number: 11415-EN BACKGROUND CD73, known as ecto-5'-Nucleotidase, converts extracellular nucleoside 5' monophosphates to nucleosides, with AMP as its preferred substrate (1). CD73 is a zinc-dependent, 70 kDa homodimeric enzyme bound to the cell membrane through a glycosyl phophatidylinositol (GPI) anchor. It is composed of an N-terminal domain containing metal binding sites linked via small hinge region to a C-terminal domain containing the substrate binding site and dimerization interface (2). It is expressed by most cell types (3)...