2.5. Lunasin Purification Anion Exchange Chromatography

Lunasin extract (LE) was obtained as reported previously [26] with slight changes. Briefly, freeze-dried lunasin enriched soy extract (40% w/v) was solubilized in 50 mL distilled water, centrifuged twice at 12,000× g for 10 min, and filtered through a 0.45 µm filter. A HiTrap Q HP (GE Healthcare Bio-Sciences, Uppsala, Sweden) column was used coupled with a HiPrepp 26/10 desalting pre-column (GE Healthcare Bio-Sciences, Uppsala, Sweden). Unbound proteins eluted with Tris-HCl 20 mM, pH 7.4 at a 1 mL/min flow rate. Bound proteins eluted using 0.4 M NaCl. The LE sample was desalted using ultrafiltration through a 1 kDa disc and freeze-dried for in vitro analysis.