Recombinant Human Kallikrein 4/Prostase Protein, CF

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

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
• Assay Buffer: 50 mM Tris, pH 9.0
• Recombinant Human Kallikrein 4/Prostase/EMSP1 (rhKLK4) (Catalog # 1719-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
• Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011) , 90 mM Stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhKLK4 to 200 µg/mL in Activation Buffer.
2. Dilute Thermolysin to 2 µg/mL in Activation Buffer.
3. Combine equal volumes diluted rhKLK4 and Thermolysin and incubate at 37 °C for 2 hours to activate.
4. Stop the reaction with 1,10 Phenanthroline at a final concentration of 10 mM.
5. Dilute activated rhKLK4 to 2 ng/µL in Assay Buffer.
6. Dilute Substrate to 200 µM in Assay Buffer.
7. Load 50 µL of the 2 ng/μL rhKLK4 in a 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 without any rhKLK4.
8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
9. Calculate specific activity:

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

• rhKLK4: 0.100 µg
• Substrate: 100 µM