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Comparative tissue proteomics aims to analyze alterations of the proteome in response to a stimulus. Two-dimensional difference gel electrophoresis (2D-DIGE) is a modified and advanced form of 2D gel electrophoresis. DIGE is a powerful biochemical method that compares two or three protein samples on the same analytical gel, and can be used to establish differentially expressed protein levels between healthy normal and diseased pathological tissue sample groups. Minimal DIGE labeling can be used via a 2-dye system with Cy3 and Cy5 or a 3-dye system with Cy2, Cy3, and Cy5 to fluorescently label samples with CyDye flours pre-electrophoresis. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization. This form of quantitative high-resolution proteomics facilitates the comparative analysis and evaluation of tissue protein compositions. Comparing tissue groups under different conditions is crucially important for advancing the biomedical field by characterization of cellular processes, understanding pathophysiological development and tissue biomarker discovery. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of 2-dye and 3-dye DIGE minimal labeling.

Original publication





Publication Date





185 - 202


2-Dye labeling, 3-Dye labeling, CyDyes, Difference gel electrophoresis, Isoelectric focusing, Mass spectrometry, Protein digestion, Protein identification, Protein separation, Tissue proteomics, Two-dimensional gel electrophoresis, Animals, Chromatography, Liquid, Fluorescent Dyes, Humans, Image Processing, Computer-Assisted, Isoelectric Focusing, Male, Mass Spectrometry, Proteome, Proteomics, Staining and Labeling, Testis, Two-Dimensional Difference Gel Electrophoresis