Recombinant Human Arginase 2/ARG2 Protein, CF

•  Assay Diluent: Deionized Water
  
• Recombinant Human Arginase 2/ARG2 (rhARG2) (Catalog # 9767-AR)
• Urea, 100 mM stock in deionized water
  
• Substrate Buffer: 125 mM L-Arginine, 625 mM Glycine, pH 10.5
• Manganese Chloride, 1 M stock in deionized water
• O-Phthaldialdehyde (oPA), (Sigma, Catalog # P0657), 50 mg/mL (373 mM)  stock in DMSO
• N-(1-Naphthyl)ethylene-diamine dihydrochloride (NED) (Sigma, Catalog # N9125), 500 mM stock in DMSO
• 50 mM Boric Acid, 1 M Sulfuric Acid, 0.03% Brij-35 (w/v) [Caution: highly acidic, neutralize before disposal]
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhARG2 to 0.8 µg/mL in deionized water.
2. Prepare a standard curve from the 100 mM Urea stock. Dilute 100 µL of 100 mM Urea with 900 µL of deionized water to make a 10 mM Urea solution. Use this for the first point of the curve.
3. Complete the standard curve by performing six one-half serial dilutions of the 10 mM Urea stock in deionized water. The standard curve has a range of 7813 to 500,000 pmol per well.
4. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of deionized water.
5. Load 25 µL of to 0.8 µg/mL rhARG2 into empty wells of the same plate as the curve. Load multiple wells as some will be used as controls.
6. From the Substrate Buffer and Manganese Chloride stocks, prepare a solution of 60 mM Arginine, 300 mM Glycine, 40 µM MnCl2, pH 10.5. Do not prepare this solution until immediately before use as the manganese will gradually precipitate out of solution.
7. Add 25 µL of 60 mM Arginine, 300 mM Glycine, 40 µM MnCl2, pH 10.5 to the wells containing 0.8 µg/mL rhARG2 (exclude the controls). Mix well. 
8. Cover the plate and incubate at 37° C for 30 minutes.
9. Dilute oPA stock to 4 mM in 50 mM Boric Acid, 1 M Sulfuric Acid, 0.03% Brij-35 (w/v).
10. Dilute NED stock to 4 mM in 50 mM Boric Acid, 1 M Sulfuric Acid, 0.03% Brij-35 (w/v).
11. Combine equal volumes of 4 mM oPA and 4 mM NED to form a solution of 2 mM oPA with 2 mM NED.
12. Add 200 µL of 2 mM oPA, 2 mM NED solution to all wells, including the standard curve.
13. Prepare a fresh solution of 60 mM Arginine, 300 mM Glycine, 40 µM MnCl2, pH 10.5. Add 25 µL to each well used as a control.
14. Cover the plate and incubate at room temperature for 20 minutes.
15. Read plate at 520 nm (absorbance) in endpoint mode.
16. Calculate specific activity:

*Derived from the urea standard curve using linear or 4-parameter fitting and adjusted for controls.

Per Reaction:

• rhARG2: 0.02 µg
• Arginine: 6 mM
• oPA & NED: 1.6 mM