Recombinant Human Cathepsin B 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 5.0
• Assay Buffer: 25 mM MES, pH 5.0
• Recombinant Human Cathepsin B (rhCathepsin B) (Catalog # 953-CY)
• Fluorogenic Peptide Substrate VII: Z-Leu-Arg-AMC (Catalog # ES008)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCathepsin B to 10 µg/mL in Activation Buffer.
2. Incubate at room temperature for 15 minutes.
3. Dilute rhCathepsin B to 0.2 ng/µL in Assay Buffer.
4. Dilute substrate to 20 µM in Assay Buffer.
5. Load 50 µL of the 0.2 ng/µL rhCathepsin B in a black well 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 without any rhCathepsin B.
6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
   Calculate specific activity:

     *Adjusted for Substrate Blank

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

• rhCathepsin B: 0.01 µg
• Substrate: 10 µM