Purification of alpha-arylphorin

Arylphorin levels are highest at the pre-wandering phase of the 5th larval instar in noctuid larvae (Burmester, 2015). Consequently, hemolymph was collected from 60 pharate 5th instar H. virescens larvae by making a small incision at the base of the 1st and/or 2nd proleg and collecting droplets into 15 mL conical tubes containing 5 mg of phenylthiourea to block hemolymph phenoloxidase activity, and maintaining on ice. After collection, hemolymph was frozen at −20 °C until used (no longer than 2 months). Frozen hemolymph was thawed on ice and diluted 5-fold in 20 mM Tris pH 7.9 (buffer A). For fractionation, hemolymph was filtered (0.22 µm) and loaded onto a HiTrap Q HP column (GE Healthcare, Little Chalfont, UK), previously equilibrated with buffer A and connected to an AKTA FPLC system (GE Healthcare, Little Chalfont, UK). Proteins were eluted with a 0–1 M linear gradient of NaCl in 20 mM Tris pH 7.9 (buffer B) at a flow rate of 1 mL/min, collecting 1 mL fractions. To reduce the presence of smaller proteins co-purifying with α-arylphorin, fractions estimated to contain α-arylphorin (based on presence of ∼70 kDa band on electrophoretic observations) were combined and filtered using an Amicon Ultra-15 mL centrifugal unit (Millipore, Billerica, MA, USA) with a MWCO of 50 kDa. After concentration, partially purified α-arylphorin (Fig. S1) was quantified with the Coomassie Plus Protein Assay (Pierce, Waltham, MA, USA) using BSA as the standard, and then aliquoted and maintained at −80 °C until used.