Recombinant Rat Cathepsin C/DPPI 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: 25 mM MES, 5 mM DTT, pH 5.5
• Assay Buffer: 25 mM MES, 50 mM NaCl, 5 mM DTT, pH 6.0
• Recombinant Rat Cathepsin C/DPPI (rrCathepsin C) (Catalog # 8285-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 rrCathepsin 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 rrCathepsin C and diluted rhCathepsin L for final concentrations of 10 µg/mL and 1 µg/mL respectively.
4. Incubate at room temperature for 30 minutes to activate rrCathepsin C.
5. Dilute activated rrCathepsin C to 0.02 µg/mL in Assay Buffer.
6. Dilute Substrate to 100 µM in Assay Buffer.
7. Load 50 µL of the 0.02 µg/mL rrCathepsin C into a plate, and start the reaction by adding 50 µL of 100 µM Substrate.  Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 100 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 # A-9891).

• rrCathepsin C: 0.001 µg
• Substrate: 50 µM