Recombinant Mouse 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 opening.

• Assay Buffer: 50 mM MES, pH 6.0
• Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock
• Recombinant Mouse Cystatin F (rmCystatin F) (Catalog # 4557-PI)
• Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
• Substrate: Z-Leu-Arg-AMC (Catalog # ES008) , 2 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 40 µg/mL in Assay Buffer with 5 mM DTT.
2. Incubate on ice for 15 minutes.
3. After incubation, dilute activated rhCathepsin L to 0.2 µg/mL in Assay Buffer.
4. Prepare a curve of rmCystatin F (MW: 15249 Da) in Assay Buffer. Make the following serial dilutions: 6000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 5.208 nM.
5. Gently mix equal volumes of the rmCystatin L 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 5 fold dilution of the incubated curve dilutions in Assay Buffer.
8. Dilute Substrate to 20 µM in Assay Buffer.
9. Load 50 µL of the diluted incubated mixtures in a 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 the 50% inhibition concentration (IC₅₀) for rmCystatin F by plotting RFU/min (or specific activity) vs. 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: 0.001 µg
• rmCystatin F curve: 300, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.781 and 0.260 nM
• Substrate: 10 µM