Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel.
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 alpha-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.
Kitta
Kazumi
K
National Food Research Institute, Research Planning and Coordination Division, Tsukuba, Ibaraki 305-8642, Japan. kaz@nfri.affrc.go.jp
Ohnishi-Kameyama
Mayumi
M
Moriyama
Tatsuya
T
Ogawa
Tadashi
T
Kawamoto
Shinichi
S
D019342:Acetic Acid; D000485:Allergens / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005512:Food Hypersensitivity; D007073:Immunoglobulin E / Q000737:chemistry; D007525:Isoelectric Focusing / Q000379:methods; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014908:Triticum / Q000276:immunology; D014508:Urea
D000485:Allergens; D010940:Plant Proteins; D007073:Immunoglobulin E; D014508:Urea; D019342:Acetic Acid