Two-Dimensional Gel Electrophoresis 2D-GE

In 2D-GE, two electrophoresis modes are combined on a single gel. One separation is performed in the first dimension, followed by another separation perpendicular to the first one. With this method, mixtures with thousands of proteins or nucleic acids can be separated with high resolution. The resulting fingerprint can be compared to electronic databases. Results can, however, be difficult to reproduce and this technically challenging method requires experienced operating personnel. 

2D-GE can be performed on small plates with an area of a few cm, but larger plates, which measure 45 x 30 cm are also commonly used. After separation, the analytes must be visualised, for example by staining. The gels are then scanned and can be compared to electronic databases. 2D-GE is commonly applied to proteins, DNA and RNA. 

2D electrophoresis of RNA usually employs a gel in native conditions in one dimension, i.e. the RNA molecules retain their native folding configuration. In the second dimension the gel contains a denaturing agent such as SDS. Thus, the RNA molecules undergo a change in conformation and can be separated from each other. 

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The group of Mann [2-3] pioneered in nanoelectrospray (nano-ESI), enabling the identification of proteins separated by two-dimensional gel electrophoresis (2D-GE), digested with trypsin, and infused by nano-ESI-MS-MS. 

For proteomics analysis, all the proteins from a cell must be extracted and then separated from each other. Gel electrophoretic methods (section 3.2) are most powerful, especially two-dimensional gel electrophoresis (2D-GE), which is capable of separating thousands of proteins in a single run (section 3.2.5). 

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. 

X-ray fluorescence is a non-destructive and multielemental analytical technique. Because of its excellent analytical sensitivity and spatial resolution under micro-beam conditions, the technique is capable of microscopic analysis, supplying information about two-dimensional (2D) distributions of trace elements. The technique can, thus, be used for imaging trace elements in biological specimens, and for the direct determination of trace elements in protein bands after slab-gel electrophoresis (GE), which is the benchmark for high-resolution protein separation, particularly in 2D format. Therefore, XRF is a useful technique for metallomics and metalloproteomics studies. 

Electrophoresis-based separation techniques have been widely used in pro-teomics studies. The most commonly used electrophoresis-based technique is gel electrophoresis (GE), which is a procedure for separating a mixture of molecules through a stationary material (gel) in an electrical field. The GE, especially two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), is a powerful tool for protein separation. ... 

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