Purification of Prophenoloxidase from H. armigera Larvae

Prophenoloxidase was purified from the hemolymph of day-3 5th instar H. armigera larvae according to the protocol reported described (30 (link)). Briefly, 10 ml hemolymph was collected from larval body and pooled into ice-cold saturated ammonium sulfate (AS). AS saturation (35-50%) was collected and loaded on column prepacked with Ceramic Hydroxyapatite (Bio-Rad, Hercules, CA, United States). The fractions with cetylpyridinium chloride (CPC) activated PO activity were combined and applied through Concanavalin A Sepharose column (Sigma-Aldrich, St. Louis, MO, United States). The flow-through fraction was applied to a Phenyl Sepharose 6 Fast Flow (low sub) column (GE Healthcare, Little Chalfont, United Kingdom). Fractions containing PO activity were applied to a Superdex 200 column (ÄKTApurifier; GE Healthcare). Purified PPO were stored at −80°C before analysis.