Biotinylated anti human ige monoclonal antibody

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of SE was performed as previously described [28] .
Briefly, for the first dimension, 80 g SE were applied using immobilized pH gradient 7 cm strips (pH 3-10); and for the second dimension, the strips were run on 12.5% (w/v) SDS-PAGE gels. Gels were stained with Coomassie blue G250. 2D-PAGE was transferred to nitrocellulose membrane and blocked with 3% horse serum in PBS. Blocked membranes were incubated with a pool of patient sera (1:5, overnight at 4ЊC), followed by biotinylated anti-human IgE monoclonal antibody (1:3000, 4 h at 37ЊC, Vector Laboratories Inc., CA, USA), and horseradish peroxidase (HRP)-streptavidin conjugate (1:3000, 30 min at 37ЊC, Sigma-Aldrich, MO, USA). Luminol chemiluminescent substrate and exposure to X-ray film (Amersham Hyperfilm ECL, GE Healthcare Bio-Sciences Corp., USA) were used for spot visualization. The protein identification of immunoreactive spots was determined by PMF MALDI-TOF MS [29] . Search parameters were set as described elsewhere (MASCOT 2.6.0, database: SwissProt 2016_10, enzyme: trypsin; variable modifications: carbamidomethyl (Cys), oxidation (Met), peptide mass tolerance: 35 ppm, taxonomy: other green plants and max missed cleavages: 2).