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Comparative 3-Sample 2D-DIGE Analysis of Skeletal Muscles

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Difference Gel Electrophoresis

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

Abstract

The skeletal muscle proteome consists of a large number of diverse protein species with a broad and dynamic concentration range. Since mature skeletal muscles are characterized by a distinctive combination of contractile cells with differing physiological and biochemical properties, it is essential to determine specific differences in the protein composition of fast, slow, and hybrid fibers. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a powerful comparative tool to analyze fiber type-specific differences between predominantly fast contracting versus slower twitching muscles. In this chapter, the application of the 2D-DIGE method for the comparative analysis of different subtypes of skeletal muscles is outlined in detail. A standardized proteomic workflow is described, involving sample preparation, protein extraction, differential fluorescence labeling using a 3-CyDye system, first-dimension isoelectric focusing, second-dimension slab gel electrophoresis, 2D-DIGE image analysis, protein digestion, and mass spectrometry.

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Acknowledgments

Research in the author’s laboratory has been supported by a project grant from the Kathleen Lonsdale Institute for Human Health Research, Maynooth University, and equipment funding under the Research Infrastructure Call 2012 by Science Foundation Ireland (SFI-12/RI/2346/3).

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

    Kay Ohlendieck

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    Kay Ohlendieck

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Ohlendieck, K. (2023). Comparative 3-Sample 2D-DIGE Analysis of Skeletal Muscles. 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_11

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  • DOI: https://doi.org/10.1007/978-1-0716-2831-7_11

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