Two-Dimensional Difference in Gel Electrophoresis for Biomarker Discovery

Abstract One of the oldest and most widely used techniques for the analysis of clinical samples for the purpose of detecting and identifying protein biomarkers is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Two-dimensional differential in-gel electrophoresis (2D-DIGE) is a novel version of 2D-PAGE that provides improved sensitivity and reproducibility. In 2D-DIGE, proteins in two different samples, along with the appropriate standards, are labeled with cyanine fluorescent dyes and spotted on the same gel. The dyes have different excitation and emission wavelengths enabling proteins within samples and standards to be detected as separate spots even though they co-migrate. Hundreds of proteins within cells, tissues, and biological fluids can be separated, detected, and quantified using this approach. Separated proteins of interest are excised, in-gel digested, and identified using mass spectrometry. The advantages and limitations of 2D-DIGE in meeting the goal of discovering novel biomarkers are discussed in this chapter.