Two-dimensional Electrophoresis

Two-dimensional (2D) polyacrylamide gel electrophoresis is a classical technique for the separation of proteins. It Rrst appeared in the mid-1970s but for a long time it only found limited applications. Recently it has enjoyed an impressive renaissance. The major reasons for this are the introduction of the immobilized pH gradient (IPG) strips and the development of analytical methods capable of identifying proteins present in very low quantities. 2D electrophoresis represents the core methodology of the new, technology-driven science proteomics. Proteomics Rnds a wide application, in both clinical diagnosis and in pharmaceutical research, for the detection of novel drug targets. Figure 1 demonstrates the application of 2D electrophoresis for the detection of variable protein levels between diseased and healthy brain tissue. In a sample from the parietal lobe of the brain of a patient with Alzheimer’s disease, a strong spot representing glial Rbrillary acidic protein (GFAP), a marker for neuronal loss, is present, whereas in the control sample, the corresponding spot is very weak. The aim of proteomics is the high throughput analysis of the proteome (protein complement expressed by a genome) of various organisms or tissues. It consists of two steps: (1) the separation of protein mixtures by 2D electrophoresis, and (2) the identiRcation of the separated proteins by analytical techniques, such as mass spectrometry and amino acid composition analysis. The process is facilitated by the use of highly sophisticated software for advanced image analysis and the high reproducibility of images in intraand inter-laboratory studies. The 2D electrophoresis itself involves: (1) separation of the proteins on the basis of differences in their net charge, called isoelectric focusing (IEF), and (2) separation of the focused proteins on the basis of differences in their molecular masses. Table 1 gives a summary of the most signiRcant chronological events in the development of 2D electrophoresis. The state-of-the-art technology will be discussed without entering into extensive technical details that can be found in the literature provided.