Simultaneous Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Analysis of Two Distinct Proteomes
This chapter describes the basics, applications, and limitations of two-dimensional gel electrophoresis (2DE) and two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of distinct proteomes. We also propose a basic protocol for 2
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Introduction It is likely that Marc Wilkins did not expect the term “proteome” that he coined during his Ph.D. in 1994 would give raise to whole science that we now know as “proteomics” [1]. In reality, proteomics had already been in action for almost 20 years before this after Patrick O’Farrell introduced a technique of protein separation in 1975 called two-dimensional gel electrophoresis (2DE) [2]. During the genome era, 2DE was the most used method for comparative global proteome analyses, combined with mass spectrometry (MS) for protein identification. Since this time, the technique has undergone many optimization steps, including the use of carrier ampholytes, immobilized pH gradients (IPG), and development of IPG acrylamide strips [3, 4]. Perhaps the most significant advancement has been the introduction of sample labeling with fluorophores, which makes 2DE more sensitive, precise, and replicable.
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_17, © Springer Science+Business Media LLC 2017
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2DE involves separation of proteins in two dimensions according to their isoelectric point (isoelectric focusing—IEF) and apparent molecular size generally by polyacrylamide gel electrophoresis (PAGE) (Fig. 1). This enables the simultaneous separation and display of hundreds of proteins on a single gel. IEF was originally performed in a capillary-gel format, with a pH gradient generated by ampholytes in a gel-matrix. These first systems had low reproducibility due to handling difficulties. As a result, IPGs were developed which are comprised of ampholytes covalently bonded to the gels. The second dimension of the method separates proteins based on their apparent molecular weight in a polyacrylamide gel matrix, in the presence of sodium dodecyl sulfate detergent (SDS). In SDS-PAGE or 1D-PAGE, large amounts of the SDS are necessary to mask the charges of the proteins via formation of anionic complexes. The electrophoretic mobility of the proteins in the polyacrylamide gel is then dependent on the molecular weight of the protein and the separation occurs when a current is applied. The reproducibility of SDS-PAGE and IEF has improved by the development of precast gels. Protein visualization or detection after 2DE separation is commonly carried out using organic dyes, silver staining, radioisotope tagging, or by fluorescent and chemiluminescent labeling agents, as discussed below [5]. After running the gels are digitalized and the resulting images can be analyzed using a variety of software. This allows comparison of protein spot densities from test and control samples across different gels. After this, spots of interest can be excised from the gel and the proteins digested for identification by MS [6, 7]. 2DE-MS remained as the main proteomic technique for many years due to advances in protein mass spectrometry, the availability of complete genome sequences, and the development of computa
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