Two dimensional electrophoresis

IPG-DALT can be used for the separation of up to 5000 components in a protein mixture, and has now been incorporated as a qualitative tool for diagnostic analysis. It is not generally used for quantitation of proteins in a mixture, however, because loss of low molecular weight components often occurs during the SDS equilibration and transfer steps. An excellent overview of the capabilities and technical protocols of IPG-DALT is available in a review article.14 

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Two-Dimensional Electrophoresis. Two-dimensional (2D) electrophoresis is unique, offering an analytical method that is both reproducible and sensitive. It is referred to as 2D because it employs two different methods of electrophoresis, in two different dimensions, to produce one result. Each method separates the sample compounds based on different properties of each compound. The combination of the two methods gives better resolution of the compounds in the sample than could be achieved with either method alone. For example, each method alone may separate up to 100 components of a sample, whereas together they may separate up to 10,000 components. 

P. H. O Eaixell, High resolution two-dimensional electrophoresis of proteins , 7. Biol. Chem. 250 4007 (1975). 

Wilkins MR et al (1996) From proteins to proteomes large scale protein identification by two-dimensional electrophoresis and amino acid analysis. BioTechnol 14 61-65... 

Harrington, MG Lee, KH Bailey, JE Hood, LE, Sponge-Like Electrophoresis Media Mechanically Strong Materials Compatible with Organic Solvents, Polymer Solutions and Two-Dimensional Electrophoresis, Electrophoresis 15, 187, 1994. 

Torres, A. R., Krueger, G. G., and Peterson, E. A., Purification of Gc-2 globulin from human serum by displacement chromatography a model for the isolation of marker proteins identified by two dimensional electrophoresis, Anal. Biochem., 144, 469, 1985. 

The difficulty with protein arrays is that proteins do not behave as uniformly as nucleic acid. Protein function is dependent on a precise, and fragile, three-dimensional structure that may be difficult to maintain in an array format. In addition, the strength and stability of interactions between proteins are not nearly as standardized as nucleic acid hybridization. Each protein-protein interaction is unique and could assume a wide range of affinities. Currently, protein expression mapping is performed almost exclusively by two-dimensional electrophoresis and mass spectrometry. The development of protein arrays, however, could provide another powerful... 

Anderson, N. G., and Anderson, N. L. (1996). Twenty years of two-dimensional electrophoresis past, present and future. Electrophoresis 17, 443-453. 

Gauss, C., Kalkum, M., Lowe, M., Lehrach, H., and Klose, J. (1999). Analysis of the mouse proteome. (I) Brain proteins Separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 20, 575-600. 

Gorg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., and Weiss, W. (2000). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037-1053. 

Rabilloud, T., Valette, C., and Lawrence, J. J. (1994). Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients. Electrophoresis 15, 1552-1558. 

Herbert, B. Advances in protein solubilisation for two-dimensional electrophoresis. Electrophoresis 1999,20, 660-663. 

Molloy, M. P. Herbert, B. R. Williams, K. L. Gooley, A. A. Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis. Electrophoresis 1999, 20, 701-704. 

Chevallet, M. Santoni, V. Poinas, A. Rouquie, D. Fuchs, A. Kieffer, S. Rossignol, M. Lunardi, J. Garin, J. Rabilloud, T. New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 1998,19,1901-1909. 

Zuo, X., Speicher, D. W. (2000). A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. Anal Biochem. 284(2), 266-278. 

High-efficiency separations of FQ-labeled proteins are only achieved in the presence of an anionic surfactant, such as SDS. As a result, capillary isoelectric focusing is not useful for the analysis of these proteins. Instead, we employ capillary sieving electrophoresis and micellar electrokinetic capillary chromatography for our two-dimensional electrophoresis. 

Hanash, S.M. (2000). Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients current status. Electrophoresis 21, 1202-1209. 

Seow TK et al. Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by... 

Araki N et al. Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis. Electrophoresis 2000 21 1880-1889. 

Journet A et al. Towards a human repertoire of monocytic lysosomal proteins. Electrophoresis 2000 21 3411-3419. Soskic V et al. Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor. Biochemistry 1999 38 1757-1764. Thiede B et al. A two dimensional electrophoresis database of a human Jurkat T-cell line. Electrophoresis 2000 21 2713-2720. 

Newsholme SJ et al. Two-dimensional electrophoresis of liver proteins characterization of a drug-induced hepatomegaly in rats. Electrophoresis 2000 21 2122-2128. 

Protein (as pure as possible) is specifically cleaved with a protease under defined conditions. Thus the mixture of peptides, specific for the given protein, is produced. In classical proteomic approach this step is generally preceded by separation of individual proteins, usually by two-dimensional electrophoresis. 

Dunbar, B.S. (1987) Two-Dimensional Electrophoresis and Immunological Techniques. Plenum, New York. pp. 229-335. 

Non-denaturing gel electrophoresis Denaturing (SDS) gel electrophoresis Two-dimensional electrophoresis Capillary electrophoresis Peptide mapping HPLC (mainly RP-HPLC)... 

Two-dimensional electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with separation in the second dimension being undertaken in the presence of SDS, thus promoting band separation on the basis of protein size. Modified electrophoresis equipment that renders two-dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-cal finished products to such systems allows rigorous analysis of purity. 

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