Recombinant Human Testican 1/SPOCK1 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, 5 mM DTT, pH 6.0
• Recombinant Human Testican 1/SPOCK1 (rhTestican 1) (Catalog # 2327-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 1 µg/mL in Assay Buffer.
2. Incubate diluted rhCathepsin L on ice for 15 minutes.
3. Dilute incubated rhCathepsin L to 0.25 µg/mL in Assay Buffer.
4. Prepare a curve of rhTestican 1 (MW: 47,800 Da) in Assay Buffer. Make the following serial dilutions: 500, 250, 125, 83.3, 55.6, 37, 24.7, and 12.3 nM.
5. Combine equal volumes of diluted rhCathepsin L and rhTestican 1 at each concentration of the curve. Include two controls containing equal volumes of Assay buffer and diluted rhCathepsin L without any rhTestican 1.
   Incubate mixtures at 37 ˚C for 15 minutes.
6. Dilute the reaction mixtures 1/5 in Assay Buffer.
7. Dilute Substrate to 20 µM in Assay Buffer.
8. Load 50 µL of the incubated mixtures in a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
9. 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% inhibition concentration (IC₅₀) value for rhTestican 1 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 (AMC) (Sigma, Catalog # A-9891).

• rhCathepsin L: 0.00125 µg
• rhTestican 1 curve: 25, 12.5, 6.25, 4.165, 2.78, 1.85, 1.235, and 0.615 nM
• Substrate: 10 µM