Complete mini proteinase inhibitor cocktail tablets

Protein was extracted from eggs on different days post-oviposition. Egg samples were homogenized using an automill (Tokken) and then suspended in PBS treated with Complete Mini Proteinase Inhibitor Cocktail Tablets (Roche, Mannheim, Germany). The egg samples were bath sonicated for 5 min, amplitude 45 (AS ONE, Osaka, Japan), and then centrifuged at 20,600 $\times$
g at 4 °C for 5 min, wherein the supernatant was recovered. After sonication and recovery of the supernatant, the egg protein concentration was measured using a Micro BCA kit (Thermo Scientific, Rockford, IL, USA). Equal protein concentrations were separated with 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked at 4 °C overnight with 3% skim milk in PBS containing 0.05% Tween 20 and then incubated with a primary antibody using mouse anti-GST1, -GST2, -Fer1, or -Fer2 serum (1:1000 dilution) at 37 °C for 1 h. After incubation with horseradish peroxidase–conjugated goat anti-mouse IgG (1:50,000 dilution; DakoCytomation, Glostrup, Denmark) at 37 °C for 1 h, the signal was detected using Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). It was analyzed using FluorChem FC2 software (Alpha Innotech, San Leandro, CA, USA).