Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid–urea polyacrylamide gel

Abstract

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid/urea–polyacrylamide gel electrophoresis (AU–PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU–PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3–10. AU–PAGE was performed as a second-dimensional electrophoresis and high resolution was obtained, especially for proteins below 15 kDa. For immunodetection, the proteins resolved on AU gel were transferred to a polyvinylidene difluoride membrane. The assembly of semidry electroblotting for AU gel was set reversed as for sodium dodecyl sulfate (SDS)–PAGE gel. The electroblotted membrane was immunolabeled with serum from a radio-allergosorbent test-positive individual for wheat to identify allergenic proteins. Protein spots strongly recognized by the patient’s serum were chosen for further analysis. Mass spectrometry analysis revealed that these proteins were α-amylase/trypsin inhibitors and lipid transfer protein. The system developed in this study was shown to be useful as a standard protocol for the separation of low-molecular weight proteins. Moreover, the IPG strips on which IEF was performed could be used either for SDS–PAGE or AU–PAGE by only changing equilibrating conditions, allowing for a wide range of allergen analysis.