Chapter 24 - Two-Dimensional Difference Gel Electrophoresis: Application for the Analysis of Differential Protein Expression in Multiple Biological Samples

Abstract

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a recently developed 2D gel-based proteomics technique that provides a sensitive, rapid, and quantitative analysis of differential protein expression between biological samples. Resolved, labeled proteins are then detected at appropriate excitation and emission wavelengths using a multi-wavelength fluorescence detection device and the signals compared. As well as reducing the number of gels that need to be run, differential labeling and mixing mean that samples are subjected to the same handling procedures and microenvironments during 2D separation, raising the confidence with which protein changes can be detected and quantified. The same gels are then poststained and proteins of interest are picked for MS identification. 2D-DIGE is applicable for the analysis of total cell lysates and complex protein mixtures from cultured cells, whole tissues, sorted or fractionated cells, whole organisms, cellular subfractions, or affinity-purified protein fractions. Thus, the problem of gel-to-gel variation is avoided. This type of single gel experiment is useful where only limited sample quantities are available.