Recombinant Mouse Trypsin 3/PRSS3 Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -70 °C under sterile conditions after opening.

• Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
• Assay Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 (w/v), pH 8.0
• Recombinant Mouse Trypsin 3/PRSS3 (rmTrypsin 3) (Catalog # 3565-SE)
• Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # 1585-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1,10 Phenanthroline (Sigma, Catalog # 320056) 0.6 M in DMSO
• Substrate MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002), 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. Activate rhEnterokinase with Thermolysin.
1. Dilute rhEnterokinase to 100 µg/mL in Activation Buffer.
2. Dilute Thermolysin to 3.16 µg/mL in Activation Buffer.
3. Mix equal volumes of diluted rhEnterokinase and Thermolysin.
4. Incubate at 37 °C for 30 minutes.
5. Stop the reaction by adding an equal volume of 20 mM 1,10 Phenanthroline to the reaction tube.
2. Activate rmTrypsin 3 with activated rhEnterokinase.
1. Dilute activated rhEnterokinase to 2 µg/mL in Assay Buffer.
2. Dilute rmTrypsin 3 to 200 µg/mL in Assay Buffer.
3. Mix equal volumes of activated rhEnterokinase and rmTrypsin 3.
4. Incubate at room temperature for 1 hour.
3. Dilute activated rmTrypsin3 to 0.04 µg/mL in Assay Buffer.
4. Dilute Substrate to 20 µM in Assay Buffer.
5. Load 50 µL of 0.04 µg/mL of rhTrypsin 3 into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
7. Calculate specific activity: 

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

• rmTrypsin 3: 0.002 µg
• Substrate: 10 µM