Technologies - 2d Gel Experiments

Nouri Neamati
University of Southern California
Biomedical and Biological Sciences

The most widely used technique for protein separation is two-dimensional gel electrophoresis (2DE). The first dimension separates proteins according to charge and the second according to mass. In the last two decades, 2DE has advanced to become a high-throughput method for the separation and purification of complex protein mixtures and the quantitation of proteins. However, its resolution still falls behind the demand, the reproducibility is sometimes questionable, and the analysis of 2D gel images is difficult. In addition, reliably measuring quantitative changes in expression levels of low abundance proteins or hydrophobic membrane proteins is rather difficult. This is why the integration of 2DE and liquid chromatography combined with MS based approaches is needed for proteomics to achieve its greatest power.

The fact that the dynamic range of protein abundance in a cell can span at least six orders of magnitude and most of the protein mass in a given cell comes from a minor number of proteins it is therefore necessary to fractionate the samples before application on a 2DE. A major limitation of high throughput proteomics is to separate and identify all the low abundance proteins in a given mixture of sample. Recently, numerous new technologies have been introduced to optimize practically all aspects of 2DE including protein separation, visualization, image analysis, and bioinformatics. We will discuss the new advances and future perspectives in this field.

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