Gel filtration chromatography: The >50K CgSE active fraction (6 mg) was applied to a Superdex 200 10/300 GL column (GE Healthcare, Tokyo, Japan) equilibrated with 0.15 M NaCl using a 500 μL loop and eluted with the same buffer at a rate of 0.25 mL min−1, using an AKTA explore 10S- FPLC system (GE Healthcare, Tokyo, Japan). A total of 35 tubes of 0.5 mL were collected and pooled into six molecular range fractions: F1, F2, F3, F4, F5, and F6.
Larval Settlement Assay: The Gel filtration-eluted fractions were subjected to a larval settlement assay at various extract amounts of 1 μg (n = 3), 10 μg (n = 6), and 50 μg (n = 5). Larval assay replications for each extract amount varied due to the limited protein yield from the eluted fractions. Several 6-well plates were pre-coated with the test extracts and the larval assay was carried out as described earlier. All data were presented as the mean ± standard deviation (SD).
SDS-PAGE Analysis: For SDS-PAGE analysis on the active fractions >50K CgSE, F2, and F3, 10 µg of each sample under reducing conditions were loaded in each lane on a 10% acrylamide gel for 1 h at 200 volts [79 (link)]. A pre-stained Protein Standard marker (Broad range, Bio-Rad, Hercules, CA, USA) was also loaded on the gel. Following SDS-PAGE, all the protein bands were visualized by the Stains-all staining method following the manufacturer’s instruction manual.
Larval Settlement Assay: The Gel filtration-eluted fractions were subjected to a larval settlement assay at various extract amounts of 1 μg (n = 3), 10 μg (n = 6), and 50 μg (n = 5). Larval assay replications for each extract amount varied due to the limited protein yield from the eluted fractions. Several 6-well plates were pre-coated with the test extracts and the larval assay was carried out as described earlier. All data were presented as the mean ± standard deviation (SD).
SDS-PAGE Analysis: For SDS-PAGE analysis on the active fractions >50K CgSE, F2, and F3, 10 µg of each sample under reducing conditions were loaded in each lane on a 10% acrylamide gel for 1 h at 200 volts [79 (link)]. A pre-stained Protein Standard marker (Broad range, Bio-Rad, Hercules, CA, USA) was also loaded on the gel. Following SDS-PAGE, all the protein bands were visualized by the Stains-all staining method following the manufacturer’s instruction manual.