Purification of Bovine Lactoferrin and Lactoperoxidase

Both bovine LP and LF were purified in our lab as previously described by Abu-Serie and El-Fakharany^{40 (link)}. In brief, bovine skim milk was obtained by defatting and decaseination of raw milk via centrifugation at 5000 rpm for 30 min and decreasing the pH to 4.2 with 1 M HCl solution, respectively^{87 (link),88 (link)}. The supernatant of skim milk was dialyzed against 50 mM Tris HCl buffer, pH 8.0. The skim milk was applied into the pre-equilibrated Mono S 5/50 GL column with 50 mM Tris HCl buffer, pH 8.0. Both LP and LF were eluted with the same buffer containing 0.0–1.0 M NaCl. The pooled fractions of LP or LF were dialyzed in 50 mM phosphate buffer pH, 7.2 overnight and applied into a Sephacryl S100 column (5 × 150 mm, GE Health care, Sweden) equilibrated with 50 mM phosphate buffer pH, 7.2 containing 150 mM NaCl and eluted with the same buffer. The homogeneity and purity of both LP and LF were evaluated by 12% SDS-PAGE. All fractions of the purified LP and LF were pooled separately, dialyzed, lyophilized, and kept at − 80 °C for further uses.