Alkaline phosphate conjugated goat anti human ige

Pollen extracts (20 µg/well) were run on a 18% SDS-PAGE gel under reducing conditions and stained with Coomassie Brilliant Blue R-250 or transferred to a polyvinylidene difluoride (PVDF) membrane (GE Waters and Process Technologies, Trevose, PA, USA). After blocking with 3% skim milk in PBST, IgE-reactive components were probed overnight using 1:4-diluted pooled serum from the 7 patients. IgE-bound proteins were detected using alkaline phosphate-conjugated goat anti-human IgE (1:1000) (Sigma-Aldrich) for 1 h, and color was developed with 1-Step^TM NBT/BCIP Substrate Solution (Thermo Fisher Scientific, Rockford, IL, USA).

Pollen extracts (20 μL each) were separated on 15%, for ragweed, mugwort, and sawtooth oak, and 18%, for Japanese hop, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. After the separation of proteins, gels were stained with Coomassie blue or transferred onto polyvinylidene difluoride membranes (GE Waters & Process Technologies, Trevose, PA, USA). IgE reactive components were detected with pooled patients’ sera (1:4, n = 10) and alkaline phosphate conjugated goat anti-human IgE (1:1,000) (ε-chain specific, Sigma-Aldrich). The color was developed with nitroblue tetrazolium and 3-bromo-4-chloro-5-indolyl-phosphate (Promega, Madison, WI, USA).

The lyophilized extract was reconstituted in distilled water or buffer, and the protein concentration was determined via Bradford assay (Bio-Rad, Hercules, CA, USA). Thereafter, the extract was aliquoted and stored at -70℃ until use. The protein profile and IgE reactive components were examined by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Lyophilized extracts were reconstituted in a solution containing 0.9% NaCl and 0.03% human serum albumin. Extracts (30 µg of protein each) were separated onto 15% gels under reducing conditions. Proteins were stained with Coomassie blue or transferred onto polyvinylidene difluoride (PVDF) membrane (0.45 µm, Millipore). Then, the membrane was incubated with 1:4 diluted sera (pooled serum from eight subjects or healthy controls). IgE antibodies were probed with alkaline phosphate-conjugated goat anti-human IgE (1:1000; Sigma-Aldrich, St. Louis, MO, USA) for 1 hour, and the color was developed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Promega, Madison, WI, USA).