2D electrophoresis

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. 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

A number of affinity-based or chromatography methods have been used to prefractionate protein samples for 2D electrophoresis. For example, proteins of low abundance can be enriched from crude lysates by affinity-... 

The utility of protein expression mapping using 2D gel electrophoresis and mass spectrometry has been demonstrated for several experimental systems. One application has been to assess the differences in protein expression between normal and cancerous cells. For example, expression mapping has been used to identify protein markers for bladder cancer (Ostergaard et al., 1999). This was accomplished by identifying proteins released into the urine of patients with and without bladder cancer using 2D electrophoresis and mass spectrometry. 

This effect is illustrated in Fig. 17.1. Multidimensional chromatography separations can be done in planar systems or coupled-column systems. Examples of planar systems include two-dimensional thin-layer chromatography (TLC) (Consden et al., 1944 Grinberg et al., 1990), where successive one-dimensional TLC experiments are performed at 90° angles with different solvents, and 2D electrophoresis, where gel electrophoresis is run in the first dimension followed by isoelectric focusing in the second dimension (O Farrell, 1975 Anderson et al., 1981 Celis and... 

Hamdan, M.H., and Righetti, P.G. (2005) Proteomics Today Protein Assessment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis, and Microarray Technology (Wiley-lnterscience Series on Mass Spectrometry), Wiley-lnterscience, ISBN-13 978-0471648178. 

Description of LC-MS, including free articles. This site has subdivisions such as proteomics, metabonomics, 2D-electrophoresis, and sample preparation. 

MAIZE-2DPAGE Maize genome 2D electrophoresis database (Maize-2DPAGE)... 

Proteins can be separated using the 2D electrophoresis method. The first dimension is separation according to the pH of the proteins. The proteins are placed on a gel strip in a buffer solution. An electrical current is applied and the proteins separate and migrate to their isoelectric points (pl)-... 

Two-dimensional-liquid chromatography (2D-LC) approaches are much easier to automate than 2D-electrophoresis. However 2D electrophoresis has the advantage that separation is performed at the protein and not at the peptide level and... 

Key Words Western blotting immunoblotting electrophoresis 2D electrophoresis immunoaffinity identification immunoblotting techniques. 

When the area of the membrane containing the protein of interest is known, another procedure, described by Lindahl (91), can be used. Only a limited area of the nitrocellulose containing the proteins is cut out and incubated with antibodies. The rest of the membrane is stained with one of the methods described in Note 10 (a method not compatible with immunodetection, but much more sensitive than Ponceau S, can be used). Thus the protein spots can be located in the 2D electrophoresis pattern, matched with a corresponding silver stained gel and translated into the protein pattern. This method also allows a considerable saving of antibodies. 

First Siena conference. 2D Electrophoresis From Protein Maps to Genomes, 5-7 September 1994. 

FIGURE 6.45 A PMMA chip for 2D electrophoresis. The credit-card-sized chip (85 mm x 55 mm x 2 mm) contains a cavity for the first-dimensional IEF channel and two buffer reservoirs for electrodes, respectively. These buffer reservoirs are physically connected by 300 parallel second-dimensional channels (50 pm x 50 pm cross section, 64 mm long) for SDS-CGE. The cavity for IEF and the CGE channels are connected to each other by an opening of 50 pm width [214], Reprinted with permission from The Royal Society of Chemistry. 

Progress in functional genomics is related to combining its methods with applications for automatic analysis of protein enzymatic activity and occurrence, accompanied by detection of protein products by means of 2D electrophoresis and mass spectrometry. This will enable detection of mRNA and protein as gene expression product, as well as comprehensive research of the relationship between genetic polymorphism, and individual and environmental factors (such as allergen or treatment exposure). 

Material collection -> 2D electrophoresis —> Protein sequencing —> Data mining tools <-> Drug Discovery... 

Currently available reagent kits, which help to remove impurities (such as nucleic acids, lipids, and carbohydrates) but which do not modify properties of proteins while detecting even trace amounts of proteins in samples, enable analysts to obtain pure proteins. Such kits are often used to prepare proteins for their separation using 2D electrophoresis, which is typically a preliminary step in the proteomics research methods. 

Figure 8.9. Resolution of proteins from heart muscle by 2D electrophoresis isoelectric focusing (IEF) x gel electrophoresis (GE). Sample size, lmg wet weight silver stain. (Courtesy of E. Jellum, University of Oslo.)...

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