Recombinant Human 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
• Recombinant Human Cathepsin H (rhCathepsin H) (Catalog # 7516-CY)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• Phosphoramidon (Tocris, Catalog # 6333), 20 mM stock in methanol
• Substrate: Arg-7-amino-4-methylcoumarin (R-AMC) (ChemImpex, 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 rhCathepsin 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 rhCathepsin H.
4. Incubate at RT for 3 hours.
5. Stop reaction by adding an equal volume of 2 mM Phosphoramidon in Assay Buffer to reaction mixture.
6. Incubate at RT for 10 minutes.
7. Add an equal volume of Assay Buffer containing 20 mM DTT to reaction mixture. The concentration of rhCathepsin H is now 25 µg/mL.
8. Incubate reaction at RT for 5 minutes.
9. Dilute activated rhCathepsin H to 1 µg/mL in Assay Buffer.
10. Dilute Substrate to 200 µM in Assay Buffer.
11. In a plate, load 50 µL of 1 µg/mL rhCathepsin 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-MethylCoumarin (AMC) (Sigma, Catalog # 9891).

Per Well:

• rhCathepsin H: 0.05 µg
• Substrate: 100 µM