Recombinant Human PSMA/FOLH1 Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM HEPES, 0.1 M NaCl, pH 7.5
• OPA Buffer: 0.2 M NaOH containing 0.1% beta -Mercaptoethanol (v/v)
• Recombinant Human PSMA/FOLH1/NAALADase1 (rhPSMA) (Catalog # 4234-ZN)
• Substrate: Ac-Asp-Glu  (Sigma, Catalog # A5930), 10 mM stock in 40 mM NaOH
• ortho-phthaldialdehyde (OPA) (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 rhPSMA to 0.4 µg/mL in Assay Buffer.
2. Dilute Substrate to 40 µM with Assay Buffer.
3. Combine 125 µL of 0.4 µg/mL rhPSMA and 125 µL of 40 µM Substrate. For a control, inactivate 125 µL of 0.4 µg/mL rhPSMA by heating it at 95 °C for 5 minutes, then add 125 µL of 40 µM Substrate Substrate.
4. Incubate at 37 °C for 1 hour.
5. Stop the reaction by heating at 95 °C for 5 minutes, then cool to room temperature.
6. Prepare a 15 mM OPA solution in OPA Buffer.
7. Add 250 µL of OPA solution to all vials and vortex.
8. Incubate at room temperature for 10 minutes.
9. Load 200 µL of reaction and control to plate.
10. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively in endpoint mode.
11. Calculate specific activity:

     *Adjusted for Substrate Blank

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

• rhPSMA: 0.020 µg
• Substrate: 10 µM
• OPA: 7.5 mM