Recombinant Human Plasma Kallikrein/KLKB1 Protein, CF

• Activation Buffer: 100 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5
• Assay Buffer:  50 mM Tris, 250 mM NaCl, pH 7.5
• Recombinant Human Plasma Kallikrein/KLKB1 (rhKLKB1) (Catalog # 2497-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• EDTA  (Sigma, Catalog # E-4884)
• Substrate Pro-Phe-Arg-AMC (Bachem, Catalog # I-1295)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhKLKB1 to 200 µg/mL in Activation Buffer.
2. Dilute Thermolysin to 20 µg/mL in Activation Buffer.
3. Combine equal volumes of 200 µg/mL rhKLKB1 and 20 µg/mL Thermolysin for final concentrations of 100 µg/mL and 10 µg/mL respectively.
4. Incubate at 37 °C for 30 minutes.
5. Stop the reaction with 50 µM EDTA.
6. Dilute incubated rhKLKB1 to 0.5 ng/µL in Assay Buffer.
7. Dilute Substrate to 200 µM in Assay Buffer.
8. Load 50 μL of the 0.5 ng/µL rhKLKB1 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-methylcoumarin (Sigma, Catalog # A9891).

Per Well:

• rhKLKB1: 0.025 µg
• Substrate: 100 µM