Recombinant Human NAALADase-2 Protein, CF

• Assay Buffer: 50 mM HEPES, 0.1 M NaCl, pH 7.5
• o-PA Buffer: 0.2 M NaOH, 0.1% 2-mercaptoethanol (v/v)
• Recombinant Human NAALADase‑2/NAALAD2 (rhNAALAD2) (Catalog # 7658-ZN)
• Substrate: Ac-Asp-Glu (Sigma, Catalog # A5930), 10 mM stock in 40 mM NaOH
• o-pthaldialdehyde (o-PA) (Sigma, Catalog # P0657), 50 mg/mL (373 mM) stock in DMSO
• 16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Dilute rhNAALAD2 to 0.5 µg/mL in Assay Buffer.
2. Dilute Substrate to 40 µM in Assay Buffer.
3. In reaction tube, mix 125 µL of rhNAALAD2 and 125 µL of Substrate. For a Control (no enzyme activity), deactivate 125 µL of rhNAALAD2 by heating it at 95-100 °C for 5 minutes, then add 125 µL of Substrate.
4. Incubate the reaction tubes and Control at 37 °C for 60 minutes.
5. Stop the reaction by heating at 95-100 °C for 5 minutes, then cool to room temperature.
6. Prepare a 15 mM o-PA solution in o-PA Buffer.
7. Add 250 µL of o-PA solution to each reaction and blank. Vortex and incubate at room temperature for 10 minutes.
8. Remove two 200 µL aliquots from each reaction and blank and transfer to the wells of a blank microplate.
9. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
10. Calculate specific activity:

     *Adjusted for Control
     **Derived using calibration standard L-Glutamic Acid (Sigma, Catalog # G8415).

• rhNAALAD2: 0.025 µg
• Substrate: 10 µM
• o-PA: 7.5 mM