Recombinant Human 5'-Nucleotidase/CD73 Protein, CF

• Assay Buffer: 25 mM Tris, 5 mM MgCl₂, pH 7.5
• Recombinant Human 5’-Nucleotidase/CD73 (rhCD73) (Catalog # 5795-EN)
• Substrate: Adenosine monophosphate (AMP) (Sigma, Catalog # A1752), 5 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

1. Dilute rhCD73 to 0.04 µg/mL in Assay Buffer.
2. 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).
3. 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.
4. Load 25 µL of 0.04 µg/mL rhCD73, standard curve, and blanks (Assay Buffer) into a plate.
   Dilute Substrate to 100 µM in Assay Buffer.
5. Add 25 µL of the Substrate to all wells. Mix well.
6. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 20 minutes.
7. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
8. Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
9. Read plate at 620 nm (absorbance) in endpoint mode.
10. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank.

Per Well:

• rhCD73: 0.001 µg
• Substrate: 35.7 µM