Recombinant Mouse Cathepsin C/DPPI Protein, CF

• Activation Buffer: 25 mM MES, 5 mM DTT, pH 5.5
• Assay Buffer: 25 mM MES, 50 mM NaCl, 5 mM DTT, pH 6.0
• Recombinant Mouse Cathepsin C/DPPI (rmCathepsin C) (Catalog # 1034-CY)
• Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
• Substrate: Gly-Arg-AMC (Bachem, Catalog # I-1215)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmCathepsin C to 20 µg/mL in Activation Buffer.
2. Dilute rhCathepsin L to 2 µg/mL in Activation Buffer.
3. Combine equal volumes of diluted rmCathepsin C and diluted rhCathepsin L for final concentrations of 10 µg/mL rmCathepsin C and 1 µg/mL for rhCathepsin L.
4. Incubate at room temperature for 20 minutes to activate rmCathepsin C.
5. Dilute activated rmCathepsin C to 0.04 ng/µL in Assay Buffer.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. Load 50 µL of the 0.04 ng/µL rmCathepsin C in a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 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 (Sigma, Catalog # A9891).

• rmCathepsin C: 0.002 µg
• Substrate: 10 µM