Recombinant Mouse Testisin/Prss21 Protein, CF

• Assay Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, pH 7.5 (TCN)
• Recombinant Mouse Testisin/Prss21 (rmTestisin) (Catalog # 6820-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1, 10-phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
• Substrate: Pro-Phe-Arg-AMC (Bachem, Catalog # I-1295), 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 rmTestisin to 200 ng/μL in Assay Buffer.
2. Dilute Thermolysin to 0.2 μg/mL in Assay Buffer.
3. Combine equal volumes of the diluted rmTestisin and diluted Thermolysin and incubate at 37 °C for 30 minutes to activate rmTestisin.
4. After incubation, stop reaction by adding an equal volume of 20 mM 1, 10-phenanthroline for a final concentration of 10 mM.
5. Dilute activated rmTestisin to 1 ng/μL in Assay Buffer.
6. Dilute Substrate to 200 μM in Assay Buffer.
7. Load into a plate 50 μL of 1 ng/μL activated rmTestisin, and start the reaction by adding 50 μL of 200 μM Substrate. For a Substrate Blank, load 50 μL of Assay Buffer and 50 μL of 200 μM Substrate.
8. Read plate at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes.
9. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A9891)

Per Well:

• rmTestisin: 0.050 μg
• Substrate: 100 μM