Recombinant Human Pepsinogen A5/PGA5 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.

• Activation Buffer: 50 mM Sodium Citrate, pH 2.5
• Assay Buffer: 50 mM Sodium Citrate, 500 mM NaCl, 0.02% Triton® X-100, pH 4.0
• Recombinant Human Pepsinogen A5/PGA5 (rhPGA-5) (Catalog # 8457-AS)
• Substrate MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002), 2 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Dilute rhPGA-5 to 100 µg/mL in Activation Buffer.
2. Incubate for 15 minutes at room temperature.
3.  Dilute activated rhPGA-5 to 0.02 µg/mL in Assay Buffer.
4. Dilute Substrate to 20 µM in Assay Buffer.
5. Load 50 µL of 0.02 µg/mL of rhPGA-5 into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
7. Calculate specific activity:

    *Adjusted for Substrate Blank
    **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975). Per Well:

• rhPGA-5: 0.001 µg
• Substrate: 10 µM