LPS binding assay

The binding of KLK peptide to LPS was determined using a Thermo Scientific™ Pierce™ LAL chromogenic endotoxin quantitation kit. Briefly, 25 μL of the KLK peptide was added in duplicate to 25 μL of Escherichia coli 0111:B4 LPS (1.0 EU/mL final concentration) for 5 and 30 min at 37°C, followed by incubation with 50 μL of Limulus Amebocyte Lysate for 10 min at 37°C. Then, 100 μL of the chromogenic substrate; Ac-Ile-Glu-Ala-Arg-p-Nitroaniline was added. After incubation at 37°C for 6 min, the reaction was stopped by the addition of 50 μL of 25% acetic acid and the OD read at 405 nm using a microplate reader. The amount of non-bound LPS was extrapolated from a standard curve, and the percentage of peptide binding to LPS calculated.