Recombinant Mouse PSMA/FOLH1 Protein, CF

• Assay Buffer: 50 mM HEPES, 0.1 M NaCl, pH 7.5
• o-PA Buffer: 0.2 M Sodium Hydroxide containing 0.1% mercaptoethanol (v/v)
• Recombinant Mouse PSMA/FOLH1/NAALADase I (rmNAALADase I) (Catalog # 4946-ZN)
• Substrate: Ac-Asp-Glu (Sigma, Catalog # A5930), 10 mM stock in 40 mM Sodium Hydroxide
• o-phthaldialdehyde (o-PA) (Sigma, Catalog # P0657) - 50 mg/mL (0.373 M) stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmNAALADase I to 0.2 µg/mL in Assay Buffer.
2. Dilute Substrate to 40 µM with Assay Buffer.
3. In reaction tube, mix 125 µL of rmNAALADase-1 and 125 µL of Substrate. For a Substrate Blank (no enzyme activity control), deactivate 125 µL of rmNAALADase-1 by heating it at 95 °C for 5 minutes, then add 125 µL of Substrate.
4. Incubate the reaction tubes and blank control at 37 °C for 60 minutes.
5. Stop the reaction tubes by heating at 95 °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 control and transfer to the wells of a black 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 Substrate Blank
     **Derived using calibration standard L-Glutamic Acid (Sigma, Catalog # G8415).

• rmNAALADase I: 0.010 µg
• Substrate: 10 µM
• o-PA: 7.5 mM