Recombinant Mouse 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), 1 M stock in deionized water
• Recombinant Mouse Cystatin E/M (rmCystatin E/M) (Catalog # 1284-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. Dilute activated Papain to 2 ng/µL in Assay Buffer.
5. Prepare a curve of rmCystatin E/M (MW: 18,954 Da) in Assay Buffer. Make the following serial dilutions:  2000, 1000, 500, 250, 125, 93.75, 62.5, 46.875, 31.25, 15.63, 7.8, and 3.9 nM.
6. Combine equal volumes of the rmCystatin E/M curve dilutions and 2 ng/µL active Papain. Include a control (in duplicate) containing equal volumes of Assay Buffer and 2 ng/µL Papain without adding any rmCystatin E/M.
7. Incubate mixtures at 37 °C for 15 minutes.
8. Make a five-fold dilution of reaction mixture with Assay Buffer.
9. Dilute Substrate to 200 µM in Assay Buffer.
10. Load into a black well plate 50 µL of the incubated mixtures 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 (top read), respectively in kinetic mode for 5 minutes.
12. Derive the 50% inhibiting concentration (IC₅₀) for rmCystatin E/M 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 (Sigma, Catalog # A-9891).

• Papain: 0.010 µg
• rmCystatin E/M curve: 100, 50, 25, 12.5, 6.25, 4.7, 3.1, 2.3, 1.57, 0.78, 0.39, and 0.195 nM
• Substrate: 100 µM