Recombinant Human SPINK1 Protein, CF

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

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Recombinant Human SPINK1 (rhSPINK1) (Catalog # 7496-PI)
• Trypsin (Sigma, Catalog # T-1426)
• Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute Trypsin to 0.25 µg/mL in Assay Buffer.
2. Prepare a curve of rhSPINK1 (MW: 7201 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81 and 3.91 nM.
3. Combine equal volumes of 0.25 µg/mL Trypsin with rhSPINK1 serial curve dilutions. Include two controls containing 0.25 µg/mL Trypsin with Assay Buffer.
4. Incubate reaction mixtures at room temperature for 30 minutes.
5. After incubation, dilute reaction mixtures 5-fold with Assay Buffer. (Example: 40 µL reaction mixture + 160 µL Assay Buffer).
6. Dilute Substrate to 20 µM in Assay Buffer.
7. In a plate, load 50 µL of the diluted reaction mixtures, and start the reaction by adding 50 µL of 20 µM Substrate to wells.
8. Read at excitation and emission wavelengths of 320 nM and 405 nM  (top read), respectively, in kinetic mode for 5 minutes.
9. Derive the 50% inhibiting concentration (IC₅₀) value for rhSPINK1 by plotting final concentration per well of rhSPINK1 vs. specific activity with 4-PL fitting.
10. Calculate specific activity for each point using the following formula (if needed):

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

• Trypsin: 0.00125 µg
• rhSPINK1 curve: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391 and 0.195 nM
• Substrate: 10 µM