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Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel.
Anal Biochem. 2006 Apr 15; 351(2):290-7.AB

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 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.

Authors+Show Affiliations

Kitta K
National Food Research Institute, Research Planning and Coordination Division, Tsukuba, Ibaraki 305-8642, Japan. kaz@nfri.affrc.go.jp
Ohnishi-Kameyama M
No affiliation info available
Moriyama T
No affiliation info available
Ogawa T
No affiliation info available
Kawamoto S
No affiliation info available

MeSH

Acetic AcidAllergensAmino Acid SequenceElectrophoresis, Gel, Two-DimensionalFood HypersensitivityImmunoglobulin EIsoelectric FocusingMass SpectrometryMolecular Sequence DataMolecular WeightPlant ProteinsSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationTriticumUrea

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16457770

Citation

Kitta, Kazumi, et al. "Detection of Low-molecular Weight Allergens Resolved On Two-dimensional Electrophoresis With Acid-urea Polyacrylamide Gel." Analytical Biochemistry, vol. 351, no. 2, 2006, pp. 290-7.
Kitta K, Ohnishi-Kameyama M, Moriyama T, et al. Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel. Anal Biochem. 2006;351(2):290-7.
Kitta, K., Ohnishi-Kameyama, M., Moriyama, T., Ogawa, T., & Kawamoto, S. (2006). Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel. Analytical Biochemistry, 351(2), 290-7.
Kitta K, et al. Detection of Low-molecular Weight Allergens Resolved On Two-dimensional Electrophoresis With Acid-urea Polyacrylamide Gel. Anal Biochem. 2006 Apr 15;351(2):290-7. PubMed PMID: 16457770.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel. AU - Kitta,Kazumi, AU - Ohnishi-Kameyama,Mayumi, AU - Moriyama,Tatsuya, AU - Ogawa,Tadashi, AU - Kawamoto,Shinichi, Y1 - 2006/01/13/ PY - 2005/11/17/received PY - 2005/12/15/revised PY - 2005/12/20/accepted PY - 2006/2/7/pubmed PY - 2006/6/22/medline PY - 2006/2/7/entrez SP - 290 EP - 7 JF - Analytical biochemistry JO - Anal Biochem VL - 351 IS - 2 N2 - 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. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/16457770/Detection_of_low_molecular_weight_allergens_resolved_on_two_dimensional_electrophoresis_with_acid_urea_polyacrylamide_gel_ DB - PRIME DP - Unbound Medicine ER -