Rabbit anti human ige antibody

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.

The assay protocol for mast cell degranulation was adapted from Bax et al. (38 ). RBL-SX38 cells were plated in 96-well plates at 10⁴ cells/well and incubated overnight at 37°C, 5% CO₂. The following day cells were sensitised with 200 ng/mL of IgE in RBL media (100 μL/well) for 24 h. Following washes with the assay buffer (Hank’s Balanced Salt Solution (HBSS) containing 1% bovine serum albumin), the allergen was added over a concentration range of 0.5 pM to 5 μM (100 μL/well, diluted in the assay buffer). A polyclonal rabbit anti-human IgE antibody (Dako, A0094) was used as a positive control. Assay buffer with 1% Triton X-100 (100 μL/well) was used to lyse the cells and determine the maximum degranulation. Assay buffer on its own was used (100 μL/well) as a negative control. Cells were incubated for 1 h at 37°C and then 25 μL/well of the supernatant was transferred to a black 96-well plate for a β-hexosaminidase release assay using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide substrate (Sigma-Aldrich) as described previously (38 ). The percentage of maximum degranulation was calculated and data were analysed and plotted using Prism 7 (GraphPad).