Serum IgE antibody binding to HSE
IgE antibody binding to HSE in mouse sera was analyzed by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and western blotting. Briefly, 20 μg of HSE was separated by SDS-PAGE with a NuPAGE® Novex 4%–12% Bis-Tris precast gel (Invitrogen, Carlsbad, CA, USA) under reducing conditions. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane. The membrane was blocked with PBS containing 5% skim milk and 0.05% Tween-20 for 2 h at room temperature, then separated into strips and incubated with the sera of sensitized or control mice (diluted 1:6) overnight at 4°C. Subsequently, polyvinylidene difluoride strips were washed with PBS containing 0.05% Tween-20 and incubated for 2 h at room temperature with rat-anti mouse IgE (diluted 1:1,000; Abcam, Cambridge, MA, USA). IgE-binding proteins were detected using BM Chemiluminescence Blotting Substrate (Roche), in accordance with the manufacturer’s instructions.