Recombinant Mouse Cathepsin H Protein, CF

• Activation Buffer: 50 mM MES, 10 mM CaCl₂, 150 mM NaCl, pH 6.5
• Assay Buffer: 50 mM MES, pH 6.5
• Dithiothreitol (DTT) (Sigma, Catalog # D-0632), 1 M stock in deionized water
• Recombinant Mouse Cathepsin H (rmCathepsin H) (Catalog # 1013-CY)
• Bacterial Thermolysin (Catalog # 3097-ZN)
• Phosphoramidon (Catalog # EI006), 20 mM in methanol
• Substrate: Arg-7-amido-4-methylcoumarin (R-AMC) (Chem-Impex, Catalog # 5859), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmCathepsin H to 200 µg/mL in Activation Buffer.
2. Dilute Thermolysin to 100 µg/mL in Activation Buffer.
3. Mix equal volumes of 100 µg/mL Thermolysin and 200 µg/mL rmCathepsin H.
4. Incubate at 37 °C for 1 hour.
5. Stop reaction by adding an equal volume of 2 mM Phosphoramidon in Assay Buffer to reaction mixture.
6. Incubate at room temperature for 10 minutes.
7. Then add an equal volume of Assay Buffer containing 20 mM DTT to reaction mixture. The concentration of rmCathepsin H is now 25 ng/µL.
8. Incubate reaction at room temperature for 30 minutes.
9. Dilute activated rmCathepsin H to 0.5 ng/µL in Assay Buffer.
10. Dilute Substrate to 200 µM in Assay Buffer.
11. In a plate load 50 µL of 0.5 ng/µL rmCathepsin H to wells, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
12. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
13. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891)

• rmCathepsin H: 0.025 µg
• Substrate: 100 µM