Recombinant Cynomolgus PSMA/FOLH1/NAALADase I Protein, CF

• Assay Buffer: 50 mM HEPES, 100 mM NaCl, pH 7.5
• o-PA Buffer: 0.2 M NaOH containing 0.1% 2-mercaptoethanol (v/v)
• Recombinant Cynomolgus Monkey PSMA/FOLH1/NAALADase I His-tag (rcynoPSMA) (Catalog # 10437-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 stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rcynoPSMA to 0.4 µg/mL in Assay Buffer.
2. Dilute Substrate to 40 µM in Assay Buffer.
3. Combine 125 µL of 0.4 µg/mL rcynoPSMA and 125 µL of 40 µM Substrate. For a Control, inactivate 125 µL of 0.4 µg/mL
    rcynoPSMA by heating it at 95 °C for 5 minutes, then add 125 µL of 40 µM Substrate.
4. Incubate the reaction tubes and control tubes at 37 °C for 60 minutes.
5. Stop the reaction by heating all tubes 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 control. Vortex and incubate at room temperature for 10 minutes.
8. Load 200 µL of each reaction and control 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 Control
**Derived using calibration standard L-Glutamic Acid (Sigma, Catalog # G8415)

• rcynoPSMA: 0.020 µg
• Substrate: 10 µM
• o-PA: 7.5 mM