Recombinant Human Cystatin C 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 C (rhCystatin C) (Catalog # 1196-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 C (MW: 14,709 Da) in Assay Buffer. Make the following serial dilutions: 1000 nM, 500 nM, 333 nM, 222 nM, 111 nM, 55.5 nM, 27.8 nM, 13.9 nM.
5. Dilute activated Papain to 2 µg/mL in Assay Buffer.
6. Mix equal volumes of the rhCystatin C curve dilutions and the diluted active Papain. Include a Papain control (in duplicate) containing Assay Buffer and the diluted active Papain.
7. Incubate mixtures at 37 °C for 10 minutes.
8. Dilute Substrate to 200 µM in Assay Buffer.
9. Perform a five-fold dilution of the incubated mixture of rhCystatin C and Papain with Assay Buffer.
10. Load into a black well plate 50 µL of diluted incubated mixture 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) for rhCystatin C 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: 0.010 µg
• Substrate: 100 µM
• rhCystatin C: 50 nM, 25 nM, 16.65 nM, 11.1 nM, 5.55 nM, 2.78 nM, 1.39 nM, 0.695 nM