Recombinant Mouse Kallikrein 1 Protein, CF

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
• Assay Buffer: 50 mM CHES, 250 mM NaCl, pH 10.0
• Recombinant Mouse Kallikrein 1 (rmKLK1) (Catalog # 7928-SE)
• Thermolysin (Catalog # 3097-ZN)
• 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
• Substrate: Pro-Phe-Arg-AMC (Bachem, Catalog # I-1295), 10 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 rmKLK1 to 200 µg/mL in Activation Buffer.
2. Dilute Thermolysin to 2 µg/mL in Activation Buffer.
3. Combine 20 µL of diluted rmKLK1 with 20 µL of diluted Thermolysin for final concentrations of 100 µg/mL and 1 µg/mL respectively.
4. Incubate at 37 °C for 1 hour.
5. Stop the reaction with 40 µL of 20 mM 1,10 Phenanthroline for a final concentration of 10 mM.
6. Dilute incubated rmKLK1 to 0.2 ng/µL in Assay Buffer.
7. Dilute Substrate to 200 µM in Assay Buffer.
8. Load 50 µL of the 0.2 ng/µL rmKLK1 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.
9. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
10. Calculate specific activity:

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

Per Well:

• rmKLK1: 0.01 µg
• Substrate: 100 µM