Recombinant Human Cystatin SN 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 SN (rhCystatin SN) (Catalog # 1285-PI)
• Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
• Substrate: Z-Leu-Arg-AMC (Catalog # ES008)
• 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 Activation Buffer.
2. Incubate 15 minutes on ice.
3. Dilute rhCathepsin L to 0.5 µg/mL in Activation Buffer.
4. Prepare a curve of rhCystatin SN (MW: 15,677 Da) in Assay Buffer. Make the following serial dilutions: 2000, 500, 200, 100, 66.7, 44.4, 22.2, 11.1, and 2.78 nM.
5. Gently mix equal volumes of the rhCystatin SN 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 ºC for 15 minutes.
7. After incubation, perform a 1/5 dilution of each reaction mixture with Assay Buffer.
8. Dilute Substrate to 20 µM in Assay Buffer.
9. Load into a black well plate 50 µL of the incubated mixtures, 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) value for rhCystatin SN 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: 0.0025 µg
• rhCystatin SN curve: 100, 25, 10, 5, 3.33, 2.22, 1.11, 0.555, and 0.139 nM
• Substrate: 10 µM