Goat anti rabbit immunoglobulin g igg

Parasite oocysts were disrupted by five freeze–thaw (liquid nitrogen–ice) cycles and lysed with a radioimmunoprecipitation assay (RIPA) buffer containing 1% Triton X-100 and a protease inhibitor cocktail for eukaryotes (Sigma–Aldrich, St. Louis, MO, USA) on ice overnight, followed by additional 1-h incubation at room temperature. Lysates equivalent to 1.0 × 10⁷ sporozoites/lane were heated with reducing sample buffer for 5 min at 95 °C, fractionated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The blots were blocked in 5% skim milk-TBST and probed with affinity-purified anti-GP900-C pAb (1:100 dilution in blocking buffer) or anti-GP900-N mAb (1:50 dilution in blocking buffer) for 1 h. After three washes with blocking buffer, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (i.e. goat anti-rabbit immunoglobulin G [IgG] or goat anti-mouse IgG; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and visualized using an enhanced chemiluminescence reagent (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. All procedures were conducted at room temperature unless specified otherwise.