Two-Dye Versus Three-Dye DIGE for Comparative Testis Tissue Proteomic Analysis

Abstract

The global analysis of the proteome is an important tool in cell biology. Comparative proteomic evaluations can identify and compare the composition, dynamics, and modifications between different samples. Comparing tissue proteomes under different conditions is crucial for advancing the biomedical field. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a sensitive and robust biochemical method that can compare multiple protein samples over a broad dynamic range on the same analytical gel and can be used to establish differentially expressed protein profiles between different sample groups. 2D-DIGE involves fluorescently labeling protein samples with CyDye flours, via a two-dye or a three-dye system, pre-separation by isoelectric point, and molecular weight. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization, thus enabling accurate high-resolution analysis of differences in protein abundance between samples. 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 two-dye and three-dye DIGE minimal labeling.