Mouse anti human ige

SDS-PAGE was conducted according to Laemmli et al. [33 (link)] using the XCELL Mini Cell System (Novex, San Diego, CA, US) with 12% polyacrylamide gels (Life Technologies, Carlsbad, CA, US) under reducing conditions. After electrophoresis, proteins were visualized by staining with colloidal Coomassie. Immunoblotting was carried out using the semi-dry blotting technique [34 (link)]. Proteins were transferred at 0.8 mA/cm² for 45 min onto a methanol-activated polyvinylidene fluoride (PVDF) membrane (pore size 0.45 μm, Millipore Corporation, Billerica, MA, US), which was blocked thereafter with TTBS (Tris buffered saline with Tween 20; 100 mM Tris, 100 mM NaCl, 2.5 mM MgCl₂, 0.05% Tween 20, pH 7.4) supplemented with 2.5% skimmed milk powder. Membrane strips were incubated with diluted patients’ sera (1:10 in TTBS, with 2.5% skimmed milk) or anti-oleosin antibodies [35 ] (1:5,000 in TTBS, with 2.5% skimmed milk) overnight. Detection of bound antibodies was conducted using alkaline phosphatase-conjugated secondary antibodies, mouse anti-human IgE (Pharmingen, San Diego, CA, US, 1:10,000) or goat anti-rabbit IgG (Jackson Immuno Research, West Grove, PA, US, 1:5,000), diluted in TTBS for 2 h. Immunostaining was carried out according to Blake et al. [36 (link)]. Blotted proteins were stained with 0.1% Coomassie in 50% methanol and Gold solution (Sigma Aldrich, St. Louis, MO, US).