Recombinant Human Trypsin 1/PRSS1 Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 100 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 8.0
• Recombinant Human Trypsin 1/PRSS1 (rhTrypsin 1) (Catalog # 3848-SE)
• Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # 1585-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002) 2 mM stock in DMSO
• 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Activate rhEnterokinase (see also R&D Systems, Catalog # 1585-SE).
1. Activate rhEnterokinase at 50 µg/mL with 1.58 µg/mL Thermolysin in Activation Buffer.
2. Incubate at 37 °C for 30 minutes.
3. Stop the reaction with an equal volume of 20 mM of 1,10 Phenanthroline for a final concentration of 10 mM.
2. Activate rhTrypsin 1 at 100 µg/mL with activated rhEnterokinase at 0.4 µg/mL.
1. Dilute the activated rhEnterokinase to 0.8 µg/mL in Assay Buffer.
2. Dilute rhTrypsin 1 to 200 µg/mL in Assay Buffer.
3. Mix equal volumes of 0.8 µg/mL rhEnterokinase with 200 µg/mL rhTrypsin 1.
4. Incubate reaction at room temperature for 15 minutes.
3. Dilute activated rhTrypsin 1 to 0.1 µg/mL in Assay Buffer.
4. Dilute Substrate to 20 µM in Assay Buffer.
5. In a plate load 50 µL of 0.1 µg/mL rhTrypsin 1 to wells, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL 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).

• rhTrypsin 1: 0.005 µg
• Substrate: 10 µM