Recombinant Human Kallikrein 6 Protein, CF

• Activation Buffer: 50 mM Tris, 0.05% (w/v) Brij-35, pH 8.0
• Assay Buffer: 50 mM Tris, 1.0 M Sodium Citrate, pH 7.5
• Recombinant Human Kallikrein 6/Neurosin (rhKLK6) (Catalog # 5164-SE)
• Lysyl-Endopeptidase, 20 U/mL stock in PBS
• Substrate: BOC-Gln-Ala-Arg-AMC (Catalog # ES014), 10 mM stock in DMSO
• Black 96-well Plate
• Plate Reader with Fluorescence Read Capability

1. Dilute rhKLK6 to 200 µg/mL in Activation Buffer.
2. Dilute Lysyl-endopeptidase to 2.5 mU/mL in Activation Buffer.
3. Combine and mix equal volumes of diluted rhKLK6 and diluted Lysyl-endopeptidase.
4. Incubate at room temperature for 30 minutes.
5. After incubation, dilute rhKLK6 to 0.5 µg/mL in Assay Buffer.
6. Dilute Substrate to 200 µM in Assay Buffer.
7. Load into a black well plate 50 µL of 0.5 µg/mL rhKLK6, 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).

Per Well:

• rhKLK6: 0.025 µg
• Substrate: 100 µM