Recombinant Human Kallikrein 11 Protein, CF

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
• Assay Buffer: 50 mM Tris, 1 M NaCl, 10 mM EDTA, pH 8.5
• Recombinant Human Kallikrein 11 (rhKLK11) (Catalog # 1595-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• EDTA (Sigma, Catalog # E-4884)
• Fluorogenic Peptide Substrate: Z-Lys-SBzl (Bachem, Catalog # M-1300), 10 mM stock in DMSO
• 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhKLK11 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 rhKLK11 and 20 µg/mL Thermolysin.
4. Incubate at 37 °C for 15 minutes.
5. Stop Thermolysin activity by adding EDTA to a final concentration of 10 mM.
6. Dilute incubated rhKLK11 to 2 ng/µL in Assay Buffer.
7. Prepare Substrate Mixture containing 600 μM Substrate and 200 μM DTNB in Assay Buffer.
8. Load into a clear plate 50 µL of the 2 ng/µL rhKLK11, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
9. Read at a wavelength of 405 nm (bottom read) in kinetic mode for 5 minutes.
10. Calculate specific activity:

     *Adjusted for Substrate Blank 
     **Using the extinction coefficient 13260 M^-1cm^-1 
     ***Using the path correction 0.320 cm
     Note: the output of many spectrophotometers is in mOD Per Well:

• rhKLK11: 0.1 µg
• Substrate: 300 µM
• DTNB: 100 µM