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Double One-Dimensional Electrophoresis (D1-DE) Adapted for Immunoproteomics

  • Youcef Shahali
  • Hélène Sénéchal
  • Pascal Poncet
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1871)

Abstract

The classical proteomics approach for the identification of allergen candidates consists on the separation of proteins by high-resolution two-dimensional electrophoresis (2-DE) with subsequent IgE immunoblotting and further analysis of IgE-reactive protein spots with mass spectrometry. In this approach at least two gels most be run. One gel is used for staining and the other is for immunoblotting by antibodies labeled with specific immunostains. Additional functional characterizations require either protein purification or 2-DE replicates and appear to be time- and reagent-consuming. Here we described a modified double one-dimensional electrophoresis (D1-DE) allowing the conversion of a protein spot previously visualized by 2-DE into an extended protein band. In D1-DE, the purity of the protein of interest is similar to 2-DE spots, but its abundance is many times higher than what can be found in a 2-DE single spot allowing many other functional analyses from a single D1-DE separation.

Key words

Double one-dimensional electrophoresis D1-DE 2-DE Allergens Proteomics 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Youcef Shahali
    • 1
  • Hélène Sénéchal
    • 2
  • Pascal Poncet
    • 2
    • 3
  1. 1.Razi Vaccine and Serum Research InstituteAgricultural Research, Education and Extension Organization (AREEO)KarajIran
  2. 2.Allergy and Environment Team, Biochemistry DepartmentArmand Trousseau Children Hospital (AP-HP)ParisFrance
  3. 3.Institute PasteurCenter for Innovation and Technological ResearchParisFrance

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