Recombinant Human Cystatin S 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.

• Activation Buffer: 50 mM MES, 5 mM DTT, pH 6.0
• Assay Buffer: 50 mM MES, pH 6.0
• Recombinant Human Cystatin S (rhCystatin S) (Catalog # 1296-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 1 µg/mL in Activation Buffer.
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 S (MW: 15,550 Da) in Assay Buffer. Make the following serial dilutions: ≥45.7 µM (stock concentration should be ≥ 0.711 mg/mL), 30.5, 20.3, 13.5, 9, 6, 4, and 2.68 µM.
5. Combine equal volumes of the rhCystatin S 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 37 °C for 15 minutes.
7. Perform a 1/5 dilution of each reaction mixture with Assay Buffer.
8. Dilute Substrate to 20 µM in Assay Buffer.
9. Load 50 µL of the incubated mixtures in a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
   Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
10. Derive the 50% inhibiting concentration (IC₅₀) of rhCystatin S by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
11. 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 (MW: 25524 Da): 0.0025 µg
• rhCystatin S curve: ≥2.286 µM (variable based on stock concentration), 1.525, 1.015, 0.675, 0.45, 0.3, 0.2, and 0.134 µM.
• Substrate: 10 µM