Recombinant Mouse 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 Mouse Cathepsin B (rmCathepsin B) (Catalog # 965-CY)
• Substrate: Z-Leu-Arg-AMC (Catalog # ES008), 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 B to 10 µg/mL in Activation Buffer.
2. Incubate at room temperature for 15 minutes (activation step).
3. Dilute activated rmCathepsin B to 0.2 ng/µL in Assay Buffer.
4. Dilute Substrate 20 µM in Assay Buffer.
5. In a plate load 50 µL of 0.2 ng/µL rmCathepsin B to wells, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank of 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
7. Calculate specific activity:

     *Adjusted for Substrate Blank

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

• rmCathepsin B: 0.01 µg
• Substrate: 10 µM