The NuPage Gel System (Invitrogen, Carlsbad, CA) was used for electrophoretic separation of protein samples by SDS-PAGE, in accordance with the manufacturer's instructions as previously described [24 ]. Samples contained 10 μg and 30 μg A. simplex protein for the immunoblotting and mass spectrometry experiments, respectively. Proteins were either stained with SimplyBlue™ Safe Stain (Invitrogen) and used for in-gel digestion and MS experiments, or transferred electrophoretically onto nitrocellulose membrane (Bio-Rad) in an XCell II Blot Module (Invitrogen) and used for immunostaining.
Immunoblots were developed as described before using Tris-buffered saline containing 0.1% Tween 20 (TBS-T, pH 7.6) as washing buffer and TBS-T containing 3% BSA as blocking and assay buffer [27 (link)]. After incubating at 4°C overnight with 1:20 diluted patient sera the blots were washed (3 × 15 min) and incubated subsequently with rabbit anti-human IgE antibody (1:1000; Dako, Glostrup, Denmark) and HRP-conjugated goat anti-rabbit antibody (1:5000; Zymed, San Francisco, CA) for 2h each with intermediate washing. After washing (3 × 10 min), the membrane was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Zymed) until bands of satisfactory intensity appeared (2–10 min). All washing and incubation steps were performed under gentle shaking at RT.
Immunoblots were developed as described before using Tris-buffered saline containing 0.1% Tween 20 (TBS-T, pH 7.6) as washing buffer and TBS-T containing 3% BSA as blocking and assay buffer [27 (link)]. After incubating at 4°C overnight with 1:20 diluted patient sera the blots were washed (3 × 15 min) and incubated subsequently with rabbit anti-human IgE antibody (1:1000; Dako, Glostrup, Denmark) and HRP-conjugated goat anti-rabbit antibody (1:5000; Zymed, San Francisco, CA) for 2h each with intermediate washing. After washing (3 × 10 min), the membrane was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Zymed) until bands of satisfactory intensity appeared (2–10 min). All washing and incubation steps were performed under gentle shaking at RT.