Recombinant Mouse Kallikrein 5 Protein, CF

• Assay Buffer: 50 mM Tris, 0.005% (w/v) Brij-35, pH 8.0
• Recombinant Mouse Kallikrein 5 (rmKLK5) (Catalog # 7236-SE)
• Recombinant Human Kallikrein 5 (rhKLK5) (Catalog # 1108-SE)
• Substrate: Boc-Val-Pro-Arg-AMC (Catalog # ES011)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmKLK5 to 200 µg/mL in Assay Buffer.
2. Dilute rhKLK5 to 2.02 µg/mL in Assay Buffer.
3. Combine equal volumes of 200 µg/mL rmKLK5 and 2.02 µg/mL rhKLK5.  For a control, combine equal volumes of 2.02 µg/mL rhKLK5 and Assay Buffer.
4. Incubate at 37 °C for 16 - 20 hours.
5. Dilute activated rmKLK5 to 1 µg/mL in Assay Buffer.  Perform same dilution on control.
6. Dilute Substrate to 800 µM in Assay Buffer.
7. In a plate, load 50 µL of 1 µg/mL rmKLK5 and control, and start the reaction by adding 50 µL of 800 µM Substrate to all wells.
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 (AMC) (Sigma, Catalog # A9891).

Per Well:

• rmKLK5: 0.050 µg
• Substrate: 400 µM