Recombinant Rat 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 opening.

• Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
• Assay Buffer: 50 mM Tris, pH 7.0
• Recombinant Rat Cystatin C (rrCystatin C) (Catalog # 6154-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. Dilute papain to 100 µg/mL in Activation Buffer.
2. Incubate at room temperature for 15 minutes.
3. Prepare a dilution curve of rrCystatin C (MW: 14,150 Da) in Assay Buffer. Make the following serial dilutions: 1000, 500, 250, 125, 83.3, 55.6, 37.0, 18.5, 9.26 and 4.63 nM.
4. Dilute activated papain to 2 µg/mL in Assay Buffer.
5. Mix equal volumes of the rrCystatin C curve dilutions and the diluted active papain. Include a papain control (in duplicate) containing Assay Buffer and the diluted active papain.
6. Incubate mixtures at 37 ºC for 15 minutes.
7. Dilute Substrate to 200 µM in Assay Buffer.
8. Perform a five-fold dilution of the incubated mixture of rrCystatin C and Papain with Assay Buffer.
9. Load into plate 50 µL of diluted incubated mixture, and start the reaction by adding 50 µL of 200 µM Substrate.
10. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
11. Derive the 50% inhibition concentration (IC₅₀) value for rrCystatin C by plotting RFU/min vs concentration with 4-PL fitting.
12. Calculate the specific activity for Papain at each point 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
• rrCystatin C: 50, 25, 12.5, 6.25, 4.17, 2.78, 1.85, 0.926, 0.463, and 0.2315 nM