Recombinant Human Serpin A4/Kallistatin Protein, CF

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

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
• Inhibition Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
• Assay Buffer: 50 mM CHES, 250 mM NaCl, pH 10.0
• Recombinant Human Serpin A4/Kallistatin (rhSerpin A4) (Catalog # 1669-PI)
• Recombinant Human Kallikrein 1 (rhKLK1) (Catalog # 2337-SE)
• Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
• 1,10-Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
• Substrate:  Pro-Phe-Arg-7-amido-4-methylcoumarin (Bachem, Catalog # I-1295), 10 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 rhKLK1 at 100 µg/mL with 1 µg/mL Thermolysin in Activation Buffer.
2. Incubate at 37 °C for 1 hour.
3. Add 1,10-Phenathroline at a final concentration of 10 mM to stop activation reaction.
4. Prepare a curve of rhSerpin A4 (47710 Da) in Inhibition Buffer. Make the following serial dilutions: 8000, 4000, 2000, 1000, 625, 415, 200, and 75 nM.
5. Dilute activated rhKLK1 to 5 µg/mL in Inhibition Buffer.
6. Combine 20 µL of 5 µg/mL rhKLK1 with 20 µL of rhSerpin A4 curve dilutions. Include two controls of 20 µL Inhibition Buffer with 20 µL of 5 µg/mL rhKLK1.
7. Incubate curve at 37 °C for 1 hour.
8. Dilute curve 12.5 fold by adding 460 µL of Assay Buffer to each dilution.
9. Dilute Substrate to 200 µM in Assay Buffer.
10. In a plate load 50 µL of the rhSerpin A4 curve, and start the reaction by adding 50 µL of 200 µM Substrate to wells.
11. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
12. Derive the 50% inhibiting concentration (IC₅₀) of rhSerpin A4 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
13. The specific activity for rhKLK1 at each point may be determined using the following formula (if needed):

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

• rhKLK1: 0.010 µg
• rhSerpin A4 curve: 160, 80, 40, 20, 12.5, 8.3, 4 and 1.5 nM
• Substrate: 100 µM