Mammalian protein extraction kit

A Mammalian Protein Extraction Kit (Solarbio, Beijing, China) was used to extract crude proteins from adult worms. Proteins were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane using an electrophoretic transfer cell (Bio-Rad, Hercules) for 30 min. After washing with TRIS-buffered saline containing Tween-20 (TBST), membranes were incubated with 5% (w/v) skimmed milk at 37 °C for 2 h and incubated at 4 °C with polyclonal antibodies (1:200 v/v dilution) or serum from infected goats (1:200 v/v dilution) for 12 h, then with a 1:1,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG (Boster, Wuhan, China) for 2 h. Signals were measured using an Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen).

A Mammalian Protein Extraction Kit (Solarbio, Beijing, China) was used to extract total protein from PSCs. Eight microliter purified rEg-IAP/rEg-BIRP proteins and 10 μL of the total protein extracts of PSCs were separated by 15% SDS-PAGE, and transferred to the nitrocellulose membranes (Millipore, Schwalbach, Germany). The membranes were blocked using 5% (w/v) skim milk at 37°C for 2 h, then incubated with CE-positive/negative sheep sera, anti-rEg-IAP/anti-rEg-BIRP rabbit IgG, or pre-immunized rabbit sera (1:200 v/v dilution) at 4°C for 12 h, separately. After four washes, membranes were incubated with horseradish peroxidase (HRP)-conjugated sheep anti-rabbit IgG or rabbit anti-sheep IgG (1:2000 v/v dilution, Bio-Rad) for 1 h at 37°C. An Enhanced HRP-DAB Chromogenic Substrate Kit (Tiangen, Beijing, China) was used for detecting the signals.