Recombinant Human Cystatin 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 reconstitution.

• Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
• Assay Buffer: 50 mM Tris, pH 7.0
• Recombinant Human Cystatin B (rhCystatin B) (Catalog # 1408-PI)
• Papain (Sigma, Catalog # P4762)
• Substrate: N-Carbobenzyloxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-AMC) (Catalog # ES009), 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. Chill Activation Buffer on ice.
2. Dilute Papain to 100 μg/mL in Activation Buffer.
3. Incubate at room temperature for 15 minutes.
4. Prepare a dilution curve of rhCystatin B (MW: 12,092 Da) in Assay Buffer. Make the following serial dilutions: 4000, 800, 400, 300, 200, 150, 100, 50, 25 and 5 nM.
5. Dilute activated Papain to 2.4 μg/mL in Assay Buffer.
6. Mix equal volumes of the rhCystatin B curve dilutions and the diluted active Papain. Include a Substrate Blank (in duplicate) containing Assay Buffer and the diluted active Papain.
7. Incubate mixtures at room temperature for 15 minutes.
8. Dilute Substrate to 200 μM in assay Buffer.
9. Perform a five fold dilution with Assay Buffer to the incubated mixture of rhCystatin B and Papain.
10. Load 50 μL of diluted incubated mixture into a plate and start the reaction by adding 50 μL of 200 μM substrate.
11. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
12. Derive the 50% inhibiting concentration (IC₅₀) value for rhCystatin B by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
13. The specific activity for Papain at each point may be determined using the following formula (if needed):

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

• Papain: .012 μg
• Substrate Z-FR-AMC: 100 μM
• rhCystatin B curve:  200, 40, 20, 15, 10, 7.5, 5, 2.5, 1.25, and 0.25 nM