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Verification of Protein Changes Determined by 2D-DIGE Based Proteomics Using Immunofluorescence Microscopy

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Difference Gel Electrophoresis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2596))

Abstract

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a key biochemical method for the comparative analysis of complex protein mixtures. The technique focuses on the identification and characterization of individual protein species following gel electrophoretic separation making it an important analytical tool of top-down proteomics. In order to verify changes in the expression levels of a particular protein, as determined by 2D-DIGE analysis, and evaluate the subcellular localization of the proteoform of interest, immunofluorescence microscopy is very well suited. This chapter describes in detail the preparation of tissue specimens and the process of cryo-sectioning, as well as incubation with primary antibodies and fluorescently labeled secondary antibodies, followed by image analysis. As illustrative examples, the co-detection of immuno-labeled dystrophin and the Y-chromosome in skeletal muscle are shown, and the localization of calbindin in the cerebellum is presented.

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Acknowledgments

We would like to thank Prof. Terry Partridge (Research Center for Genetic Medicine, George Washington University School of Medicine, Washington DC) for the generous gift of antibody p6 against dystrophin and Prof. Stephan Baader (Institute of Anatomy, University Bonn) for his support of this project.

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Authors and Affiliations

  1. Department of Neonatology and Pediatric Intensive Care, Children’s Hospital, University of Bonn, Bonn, Germany

    Margit Zweyer

  2. Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland

    Kay Ohlendieck

  3. Institute of Physiology, University of Bonn, Bonn, Germany

    Dieter Swandulla

  4. Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Bonn, Germany

    Margit Zweyer

Authors
  1. Margit Zweyer
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  2. Kay Ohlendieck
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  3. Dieter Swandulla
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Corresponding author

Correspondence to Dieter Swandulla .

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Editors and Affiliations

  1. Maynooth University, MAYNOOTH, Ireland

    Kay Ohlendieck

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Zweyer, M., Ohlendieck, K., Swandulla, D. (2023). Verification of Protein Changes Determined by 2D-DIGE Based Proteomics Using Immunofluorescence Microscopy. In: Ohlendieck, K. (eds) Difference Gel Electrophoresis. Methods in Molecular Biology, vol 2596. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2831-7_30

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  • DOI: https://doi.org/10.1007/978-1-0716-2831-7_30

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2830-0

  • Online ISBN: 978-1-0716-2831-7

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