Recombinant Mouse Hepsin 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: 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 Mouse Hepsin (rmHepsin) (Catalog # 9156-SE)
• 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 rmHepsin to 100 µg/mL in Activation Buffer.
2. Incubate at 37 °C for 24 hours.
3. Dilute activated rmHepsin to 0.2 µg/mL in Assay Buffer.
4. Dilute Substrate to 200 μM in Assay Buffer.
5. Load into a plate 50 μL of 0.2 µg/mL rmHepsin, and start the reaction by adding 50 μL of 200 μM Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 200 μM Substrate.
6. Read plate at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes.
7. Calculate specific activity:

     *Adjusted for Substrate Blank.
     **Derived using calibration standard 7-Amino, 4-Methyl Coumain (AMC) (Sigma, Catalog # A9891).

• rmHepsin: 0.010 μg
• Substrate: 100 μM