Two-dimensional gel electrophoresis 2D-PAGE

Much of the pressure to develop automated sequential HPLC separations has come from the necessity to separate complex biological mixtures, especially protein mixtures. Traditionally, complex mixtures of proteins have been separated using two-dimensional gel electrophoresis (2D GEP). The first dimension gel separation is carried out with electrophoresis buffers, the gel plate is rotated 90° and the second SDS-PAGE separation is carried out under denaturing conditions, using sodium dilauryl sulfate. The separated spots are then visualized, scraped off the plate, and then extracted for further analysis. Protein analysis by MALDI time-of-flight mass spectrometry starts with this time- and labor-intensive 2D GEP separation mode. 

Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE lEF followed by SDS-PAGE)... 

One- and two-dimensional gel electrophoresis (ID- or 2D-GE) is an important tool in the separation and isolation of intact proteins [9], In ID-GE, the proteins are separated in a sodium dodecylsulfate poly(acrylamide) gel (SDS-PAGE). The separation is according to molecular weight. In 2D-GE, the proteins are first separated by isoelectric point (pi, isoelectric focussing, lEF), and next by molecular weight. 2D-GE is considered to be the most powerful tool in protein separation. Nevertheless, the technique suffers from problems it is labour-intensive, analysis time is long, and the reproducibility poor. Furthermore, hydrophobic proteins do not behave well in the first lEF step and tend to form broad bands. 

Two Dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE) is the most powerful tool to separate the different components of complex protein mixtures. Proteins are first separated according to their p.I. in IEF (first dimension), then a further orthogonal separation according to the MW is performed by SDS-PAGE (second dimension). In this way all the protein components of a biological sample can be virtually separated. 

In proteomics the sample complexity is often high and requires high-resolution analytical separation techniques. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) fulfills this requirement and has been established as a core technique for complex protein mixtures. However, the small dynamic range and the inability to resolve very small and large proteins and proteins with extreme pi values have been serious limitations. 

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