Hrp labeled goat anti mouse igg

The silkworm samples were ground with liquid nitrogen and then lysed with RIPA (P0013B, Beyotime Biotechnology, Shanghai, China) at 4 °C for 30 min. The supernatants were separated by SDS–PAGE and transferred to a PVDF membrane (Roche, Basel, Switzerland). After blocking for 1 h at 37 °C in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20) with 5% (w/v) skim milk, membranes were incubated with 1:1000 dilutions of anti-BmUGT or negative control serum in TBST for 1 h at 37 °C. Following several washes, membranes were reacted with HRP-labeled goat anti-mouse IgG (Bio-Rad, Richmond, CA, USA), successively, with washing in between. ECL Plus Western Blotting Detection Reagents (Bio-Rad, Richmond, CA, USA) were used to detect the bound antibodies.

Spore proteins, rSWP12 and transgenic cells total protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes (Roche, Switzerland). Each membrane was blocked in 5% skimmed milk diluted in TBST (20mM Tris-HCl, 150mM NaCl, 0.05% Tween-20), incubated with mAb G4 or an anti-FLAG mouse antibody (diluted 1:3000; Sigma, Saint Louis, USA) and HRP-labeled goat anti-mouse IgG (diluted 1:6000; Bio-Rad, Richmond, California, USA), successively, with washing in between. ECL Plus Western Blotting Detection Reagents (Bio-Rad, Richmond, California, USA) were used to detect the bound antibodies. ScFv specificity was determined by immunoblots through using the scFv-G4 as primary antibody, which was extracted from the scFv-G4 expressing Sf9-III cells. The rest of the steps were same as the aforementioned protocol.