Serpin A4 Protein, Human (HEK293, His)

Materials
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
Kallikrein-1 Protein, Human (240a.a, HEK293, His) (HY-P73262)
Bacterial Thermolysin
1,10-Phenanthroline
Serpin A4 Protein, Human (HEK293, His) (HY-P71143)
Substrate: Pro-Phe-Arg-7-amido-4-methylcoumarin (PFR-AMC, HY-137784)

Procedure
1. Dilute Human KLK1 to 100 μg/mL in activation buffer (containing 1 μg/mL Bacterial Thermolysin) .
2. Incubate at 37°C for 1 hour.
3. Add 1,10-Phenanthroline to a final concentration of 10 mM to stop the activation reaction.
4. Prepare different concentrations of Human Serpin A4 in assay buffer with the following serial dilutions: 8000, 4000, 2000, 1000, 625, 415, 200, 75 nM.
5. Dilute active Human KLK1 to 5 μg/mL in inhibition buffer.
6. Mix 20 μL of 5 μg/mL Human KLK1 with 20 μL of the Human Serpin A4 dilution series. Include two controls: 20 μL of inhibitor buffer and 20 μL of 5 μg/mL Human KLK1.
7. Incubate at 37°C for 1 hour.
8. Dilute the curve 62.5 times by adding assay buffer to each dilution.
9. Dilute the substrate to 200 μM in assay buffer.
10. Add 50 μL of the diluted mixture to the plate and initiate the reaction by adding 50 μL of 200 μM substrate to the wells. For the blank control group, add 50 μL of assay buffer and 50 μL of substrate.
11. Read in kinetic mode for 5 minutes at excitation wavelength 380 nm and emission wavelength 460 nm.
12. Determine the 50% inhibitory concentration (IC₅₀) of Human Serpin A4 by plotting RFU/min versus concentration and fitting the data with a 4-parameter logistic (4-PL) curve.