Tmb stabilized substrate solution for horseradish peroxidase

To detect the expression of C1V3 protein in transgenic lines, Western blot analysis was utilized. Protein was extracted from fresh leaves in the tiller stage and dissolved in PBS buffer (pH 7.4) with protease inhibitor cocktail (MedChemExpress, Shanghai, China). Custom-made rabbit antiserum against Cry1Ab or Vip3A was used as the primary antibody, and the horseradish peroxidase-conjugated goat anti-rabbit IgG was used as the secondary antibody. TMB stabilized substrate solution for Horseradish Peroxidase (Promega, catalog number W4121) or 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich #D5905,) was used for detection. PageRuler prestained Protein Ladder (Thermo Scientific, product# 26616 & 26619) were used as marker.

Seed samples weighting 0.1 g were milled, homogenized in 1 mL PBS buffer and centrifuged at 12396 g for 10 min at 4 °C. The supernatant was collected and the expression of mAppA in the transgenic soybean was verified by Western Blot analysis. The proteins were separated by SDS-PAGE and then transferred to a PVDF membrane (Pall, Ann Arbor, MI, USA). Primary rabbit anti-mAppA polyclonal antibodies were prepared from rabbits immunized with E. coli expressing mAppA protein. The anti-mAppA antibody was then detected by horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich) as the secondary antibody. TMB stabilized substrate solution for Horseradish Peroxidase (Promega catalog number W4121) was used for detection.