Western Blot Analysis of rHc-TPS Protein

The rHc-TPS protein (30 µg) was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) as described previously (30 (link)). Afterward, the non-specific binding site by skim milk (5%) in Tris-buffered saline solution consisting of 0.1% Tween-20 solution (TBST) was blocked. Subsequently, the membranes were cleaned three times by TBST and incubated with a primary antibody (serum from goat infected with H. contortus, 1:100 dilution in TBST) at 4°C overnight. The membranes were again cleaned and incubated by HRP-conjugated rabbit anti-goat IgG (diluted 1:5,000, USA) for 1 h at 37°C. Finally, the blots were revealed by enhanced chemiluminescence (ECL) in an ImageQuant 300 cabinet (GE Healthcare Biosciences, USA) according to the manufacturer’s instructions.