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.