Differential gel electrophoresis

Second, the technology has mediocre reproducibility. Software is available to morph images so that spots can be lined up such software is expensive, difficult to use, and not always accurate in its alignment. To overcome this problem and to simplify quantitative comparisons between samples, Unlu et al. (1997) developed differential gel electrophoresis (DIGE), where two samples are each labeled with different fluorescent tags, pooled, separated on the same gel, and scanned at characteristic wavelengths to resolve the components. This technology has been commercialized by Amersham. 

For reproducible expression analysis and protein quantification MS methods based on isotopic labeling are available. They were designed in conjunction with two or more dimensional chromatographic peptide separation coupled online to MS and require advanced bioinformatics input to analyze the complex data sets in a reasonable time frame. This is also true for the alternative fluorescence-based technology of differential gel electrophoresis (DIGE Fig. 10.6) with tailor-made software which allows statistical validation of multiple data sets. 

Tonge R, Shaw J, Middleton B et al. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 2001 1 377-396. 

Beckner ME, ChenX, An J, Day BW, Pollack IF. Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies. Lab Invest 2005 85(3) 316-327. 

ZvELEBiL, M.)., Cramer, R., Waterfield, M. D., Timms, ). E. (2002). Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system. Mol. Cell Proteomics 1, 91-98. 

Rowlinson, R., Rayner, S., Young, )., PoGNAN, F., Hawkins, E., Currie, I., Davison, M. (2001). Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1, 377-396. 

Chromy BA, Gonzales AD, Perkins J, Choi MW, Corzett MH, Chang BC, Corzett CH, McCutchen-Maloney SL (2004) Proteomic analysis of human semm by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins. J Proteome Res 3 1120-1127. 

Alfonso, P., Nunez, A., Madoz-Gurpide, J., Lonbardia, L., Sanchez, L and Casal, J. I. (2005) Proteomic expression analysis of colorectal cancer by two-dimensional differential gel electrophoresis. Proteomics 5, 2602-11. 

Figure 4. DIGE (differential gel electrophoresis) analysis. A flow diagram illustrating the use of Cy3, CyS and Cy2 dyes in the analysis of control versus diseased samples. The Cy3 dye is used to label one protein sample (e.g. control sample), whilst the other is labeled with CyS (e.g. diseased/experimental sample). The labeling reaction is terminated and equal amounts of each labeled sample are combined. In parallel, equal concentrations of both control and diseased samples are pooled in one tube and labeled with Cy2. This labeled sample is used for normalisation purposes and acts as an internal standard. Both the mixture of Cy3 and CyS labeled proteins and the Cy2 labeled pooled sample are separated on the same gel. Each 2DE protein profile associated with each individual dye is visualized at a specific wavelength.

Figure 6. Differential gel electrophoresis of hepatocellular carcinoma. Cy3 (green), normal Cy5 (red), tumorous tissue.

Unlu, M. Morgan, M.E. Minden, J.S. Differential gel electrophoresis a single gel method for detechng changes in protein extracts. Electrophoresis 1997,18, 2071-2077. 

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