Recombinant Human Cystatin D Protein, CF

• Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
• Assay Buffer: 50 mM Tris, pH 7.0
• Recombinant Human Cystatin D (rhCystatin D) (Catalog # 1202-PI)
• Papain (Sigma, Catalog # P4762)
• Substrate: Z-Phe-Arg-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 D (MW: 15,273 Da) in Assay Buffer. Make the following serial dilutions: 6000, 3000, 1000, 500, 250, 100, 50, 10, and 1 nM.
5. Dilute activated Papain to 2 µg/mL in Activation Buffer.
6. Mix equal volumes of the rhCystatin D curve dilutions and the diluted active Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
7. Incubate mixtures at 37 °C 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 D curve 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. Include a Substrate Blank by combining 50 μL of 200 μM Substrate and 50 μL Assay Buffer.
11. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
12. Derive the 50% inhibition concentration (IC₅₀) for rhCystatin D by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
13. The specific activity for Papain at each point may be derived using the following formula (if needed):

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

Per Well:

• Papain: 0.010 µg
• Substrate: 100 µM
• rhCystatin D: 300, 150, 50, 25, 12.5, 5, 2.5, 0.5, and 0.05 nM