Recombinant Human PRCP Protein, CF

• Assay Buffer: 50 mM Sodium Acetate, 0.1 M NaCl, pH 4.0
• Recombinant Human Lysosomal Pro‑X Carboxypeptidase/PRCP (rhPRCP) (Catalog # 7164-SE)
• Substrate: Z-Pro-Ala (Bachem, Catalog # C-2485), 100 mM stock in deionized water
• 2-Mercaptoethanol (Sigma, Catalog # M7154)
• NaOH, 2 M stock in deionized water
• o-Phthaldialdehyde (OPA) (Sigma, Catalog # P0657), 0.373 M in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhPRCP to 0.2 µg/mL in Assay Buffer.
2. Dilute Substrate to 400 µM in Assay Buffer.
3. Mix 125 μL of 0.2 µg/mL rhPRCP and 125 μL 400 µM Substrate for a final concentration of 0.1 µg/mL and 200 µM respectively. Include a control containing 125 μL of 0.2 µg/mL rhPRCP that has been heat treated for 5 minutes at 100 °C and 125 μL 400 µM Substrate.
4. Incubate for 30 minutes at 37 °C.
5. Stop reaction by adding 250 μL of a solution containing 15 mM o-PA in 0.2 M NaOH containing 0.1% (v/v) 2-Mercaptoethanol.
6. Incubate for 10 minutes at room temperature.
7. Load 200 µL of the incubated samples in duplicate into the plate.
8. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
9. Calculate Specific Activity:

     *Adjusted for heat treated control
     **Derived using calibration standard L-Alanine (Sigma, Catalog # A7469).

• rhPRCP: 0.01 µg
• Substrate: 0.1 mM