Recombinant Human Trypsin 3/PRSS3 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, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 8.0 
• Recombinant Human Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3710-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)
• 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 with Thermolysin.
1. Dilute rhEnterokinase to 100 µg/mL with Activation Buffer.
2. Dilute Thermolysin to 3.16 μg/mL with Activation Buffer. 
3. Mix equal volumes of diluted rhEnterokinase with Thermolysin
4. Incubate at 37 °C for 30 minutes.
5. Stop the reaction by adding an equal volume of 20 mM 1,10 Phenanthroline.
2. Activate rhTrypsin 3, with activated rhEnterokinase.
1. Dilute the activated rhEnterokinase to 2 µg/mL in Assay Buffer.
2. Dilute rhTrypsin 3 to 200 µg/mL in Assay Buffer.
3. Mix equal volumes of the diluted rhEnterokinase and diluted rhTrypsin 3.
4. Incubate at 37 °C for 90 minutes.
3. After incubation, dilute activated rhTrypsin 3 to 0.1 µg/mL in Assay Buffer.
4. Dilute Substrate to 20 µM in Assay Buffer.
5. Load in a black well plate 50 µL of rhTrypsin 3 at 0.1 µg/mL, 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).

• rhTrypsin 3: 0.005 µg
• Substrate: 10 µM