Recombinant Human Cystatin E/M 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.

• Assay Buffer: 50 mM Tris, pH 7.0
• Dithiothreitol (DTT) (Sigma, Catalog # D0632)
• Recombinant Human Cystatin E/M (rhCystatin E/M) (Catalog # 1286-PI)
• Papain (Sigma, Catalog # P-4762)
• 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 the Assay Buffer on ice.
2. Dilute Papain to 100 µg/mL in Assay Buffer with 5 mM DTT.
3. Incubate at room temperature for 15 minutes.
4. After incubation, dilute activated Papain to 2.05 ng/µL in Assay Buffer.
5. Prepare a curve of rhCystatin E/M (MW: 14,953Da) in Assay Buffer. Make the following serial dilutions:  2000, 500, 250, 125, 62.5, 31.25, 15.625, 5, and 1 nM.
6. Combine equal volumes of the rhCystatin E/M curve dilutions and 2.05 ng/µL active Papain. Include a control (in duplicate) containing equal volumes of Assay Buffer and 2.05 ng/µL Papain without adding any rhCystatin E/M.
7. Incubate mixtures at 37 °C for 10 minutes.
8. Dilute the reaction mixture 5 fold with Assay Buffer.
9. Dilute Substrate to 200 µM in Assay Buffer.
10. Load 50 µL of the incubated mixtures in a plate, and start the reaction by adding 50 µL of 200 µM Substrate.
    Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in kinetic mode for 5 minutes.
11. Derive the 50% inhibiting concentration (IC₅₀) for rhCystatin E/M by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
12. The specific activity of 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 (Sigma, Catalog # A-9891).

• Papain: 0.01025 µg
• rhCystatin E/M curve: 100, 25, 12.5, 6.25, 3.125, 1.5625, 0.781, 0.25, and 0.05 nM
• Substrate: 100 µM