Recombinant Human Cystatin F 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 MES, pH 6.0
• Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
• Recombinant Human Cystatin F (rhCystatin F) (Catalog # 1889-PI)
• Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
• Substrate: Z-Leu-Arg-AMC (Catalog # ES008), 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 rhCathepsin L to 1 µg/mL in Assay Buffer with 5 mM DTT.
2. Incubate on ice for 15 minutes.
3. After incubation, dilute activated rhCathepsin L to 0.5 µg/mL in Assay Buffer.
4. Prepare a curve of rhCystatin F (MW: 15,356 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 3.9 nM.
5. Gently mix equal volumes of the rhCystatin F curve dilutions and the diluted active rhCathepsin L. Include a control (in duplicate) containing Assay Buffer and the diluted active rhCathepsin L.
6. Incubate mixtures at room temperature for 15 minutes.
7. Make a five-fold dilution of reaction mixture with Assay Buffer.
8. Dilute Substrate to 20 µM in Assay Buffer.
9. Load 50 µL of the incubated mixtures into a black well plate, and start the reaction by adding 50 µL of 20 µM Substrate.
10. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
11. Derive 50% inhibition concentration (IC₅₀) value for rhCystatin F by plotting RFU/min (or specific activity) versus concentration with 4‑PL fitting.
12. The specific activity for rhCathepsin L 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).

• rhCathepsin L: 0.0025 µg
• rhCystatin F curve: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.20 nM
• Substrate: 10 µM