Recombinant Human Hepsin Protein, CF

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

• Activation Buffer: 0.1 M Tris, 10 mM CaCl₂, 0.15 M NaCl, 0.05 % Brij-35, pH 8.0
• Assay Buffer: 50 mM Tris, pH 9.0
• Recombinant Human Hepsin (rhHepsin) (Catalog # 4776-SE)
• Fluorogenic Peptide Substrate: BOC-Gln-Arg-Arg-AMC (Bachem, Catalog # I-1655), 5 mM stock in 50:50 DMSO:Methanol
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhHepsin to 100 µg/mL in Activation Buffer.
2. Incubate at 37 °C for 24 hours.
3. Dilute activated rhHepsin to 0.2 ng/µL in Assay Buffer.
4. Dilute Substrate to 200 µM in Assay Buffer.
5. Load 50 µL of the 0.2 ng/µL rhHepsin in a black well plate and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in kinetic mode for 5 minutes.
7. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

• rhHepsin: 0.010 µg
• Substrate: 100 µM