Recombinant Human Kallikrein 14 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 Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 50 mM Tris, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 8.0
• Recombinant Human Kallikrein 14 (rhKLK14) (Catalog # 2626-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• EDTA  (Sigma, Catalog # E-4884)
• Substrate:  BOC-Val-Pro-Arg-AMC (Catalog # ES011), 100 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate reader (Model: SpectramMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhKLK14 to 200 µg/mL in Activation Buffer.
2. Dilute Thermolysin to 20 µg/mL in Activation Buffer.
3. Combine 25 µL of diluted rhKLK14 and 25 µL of diluted Thermolysin.
4. Incubate at 37 °C for 1 hour.
5. Stop reaction with 50 µL of 100 mM EDTA.
   Dilute incubated rhKLK14 mixture to 0.2 ng/µL in Assay Buffer.
6. Dilute Substrate to 200 µM in Assay Buffer.
7. Load 50 µL of the 0.2 ng/µL rhKLK14 mixture in a plate, 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.
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 # A-9891).

• rhKLK14: 0.01 µg
• Substrate: 100 µM