Recombinant Mouse Plasma Kallikrein/KLKB1 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, pH 7.5 (TCN)
• Assay Buffer: 50 mM Tris, 250 mM NaCl, 1 mM EDTA, pH 7.5
• Recombinant Mouse Plasma Kallikrein/KLKB1 (rmKLKB1) (Catalog # 2498-SE)
• Activator: Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• Deactivator: EDTA  (Sigma, Catalog # E-4884)
• Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmKLKB1 to 200 µg/mL in Activation Buffer.
2. Dilute Thermolysin to 20 µg/mL in Activation Buffer.
3. Activate rmKLKB1 by mixing equal volumes of diluted rmKLKB1 and Thermolysin together and incubate at 37 °C for 1 hour.
4. After incubation, stop reaction with 50 mM EDTA (final concentration).
5. Dilute incubated rmKLKB1 to 1 ng/µL in Assay Buffer.
6. Dilute Substrate in Assay Buffer to 20 µM.
7. In a plate load 50 µL of the 1 ng/µL rmKLKB1, and start the reaction by adding 50 µL 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 20 uM Substrate without any rmKLKB1.
8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
9. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

• rmKLKB1: 0.05 µg
• Substrate: 10 µM