Recombinant Human Tryptase gamma-1/TPSG1 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, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5 (TCNB)
• Assay Buffer: 0.1M Tris, pH 8.0
• Recombinant Human Tryptase  gamma ‑1/TPSG1 (rhTPSG1) (Catalog # 1667-SE)
• Trypsin, (Sigma, Catalog # T-1426)
• Recombinant Human Serpin A1/ alpha 1‑Antitrypsin (rhSerpin-A1) (Catalog # 1268-PI)
• 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. Dilute rhTPSG1 to 100 µg/mL in Activation Buffer.
2. Dilute Trypsin to 0.4 µg/mL in Activation Buffer.
3. Combine equal volumes of 100 µg/mL rhTPSG1 and 0.4 µg/mL Trypsin. Include a control containing Activation Buffer in place of rhTPSG1 (Trypsin control).
4. Incubate at 37 °C for 2 hours.
5. Dilute rhSerpin-A1 to 18.2 µg/mL in Assay Buffer.
6. Add rhSerpin-A1 to the reaction mixture making a one half dilution for final concentrations of 9.1 µg/mL rhSerpin-A1, 25 µg/mL rhTPSG1, and 0.1 µg/mL Trypsin. Addition of rhSerpin-A1 will stop the activity of Trypsin. Control from step three will verify that rhSerpin-A1 is working correctly.
7. Incubate all samples for 15 minutes at room temperature.
8. Dilute rhTPSG1 to 4 ng/µL in Assay Buffer. Dilute Trypsin control equally.
9. Dilute Substrate to 20 µM in Assay Buffer.
10. Load 50 µL of the diluted samples into a plate, 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.
11. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
12. Calculate specific activity:

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

• rhTPSG1: 0.200 µg
• Substrate: 10 µM