Two-dimensional gel electrophoresis. See

Analysis typically involves three steps. First, exoproteins are isolated from bacterial cultures. The aim of this process is to retain as many proteins as possible while eliminating components that inhibit isoelectric focusing. To do so, proteins are enzymatically treated and precipitated to remove salts and other non-protein components. Second, the proteins are resolved by one- or two-dimensional gel electrophoresis (see Fig. 1). Third, proteins are visualized and the composition and quantities of protein spots determined. The protocol described below uses a mini-gel format that is relatively fast and requires small culture volumes compared with large-format two-dimensional gel electrophoresis. 

Two-Dimensional Gel Electrophoresis As shown in Figure 3-22, two-dimensional gel electrophoresis allows the separation and display of up to 1,000 different proteins on a single gel. Mass spectrometry (see Box 3-2) can then be used to partially sequence individual protein spots and assign each to a gene. The appearance and nonappearance (or disappearance) of particular protein spots in samples from different tissues, from similar tissues at different stages of development, or from tissues treated in ways that simulate a variety of biological conditions can help define cellular function. 

Proteins are separated by one-dimensional, or more often, two-dimensional gel electrophoresis. The separated proteins are subjected to in situ tryptic digestion, and the peptides are separated by liquid chromatography and identified by mass spectrometry. See Nishihara, J.C. and Champion, K.M., Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain, Electrophoresis 23, 2203-2215, 2002. 

In the field of proteomics, arrays can also be used to identify possible interaction partners. Here, the array is spotted with recombinant proteins or antibodies and then hybridised with labelled cell lysate or an expressed cDNA library. Additionally, techniques like two-dimensional gel electrophoresis, the identification of isolated proteins by mass spectrometry or the two-hybrid analysis are valuable tools to identify new proteins. These methods allow a large-scale study of viral and cellular proteins without knowledge of the DNA sequence (Fig. 1, IV. For review, see Pandey and Mann 2000). 

Treatment of either a tissue culture or a cell extract with Bodipy-epoxomicrn MVB-003 followed by SDS-PAGE readily reveals proteasome active sites (Figure 12.3c) [9, 10]. In case the constitutive proteasome is expressed exclusively, the three catalytic species pi, P2, and pS are readUy resolved. In case the treated tissue also expresses immunoproteasomes, the one-dimensional gel wUl resolve pi and p2i, but only partially pi, pii, pS, and pSi (see for a detailed experimental protocol Box 12.1 [11]). Two-dimensional gel electrophoresis, by which proteins are separated on the basis of the charge followed by mass allows for complete resolution of all six catalytic activities. Figure 12.3d represents an example of competitive ABPP. In this experiment, tissue or tissue extract is treated first with a prospective inhibitor. Ensuing incubation with... 

Fig. 8,7. Routine for two-dimensional acrylamide electrophoresis, from Vigne and Jordan (1971) (for explanation see text). Other workers prefer to embed the gel strip after the first separation at the bottom of the slab, for the samples to migrate upwards...

See also-. Capillary Electrophoresis Overview. Electrophoresis Principles Isotachophoresis Isoelectric Focusing Polyacrylamide Gels Two-Dimensional Gels Blotting Techniques. 

See alsa Electrophoresis Oven/iew Isoelectric Focusing Two-Dimensional Gels Blotting Techniques Proteins. Forensic Sciences DNA Profiling. Proteins Traditional Methods of Sequence Determination. Proteomics. 

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