Gel Electrophoresis GE

Innovations in separation science continued on this theme and provided one of the most powerful separation techniques used in biochemistry, where proteins are separated with isoelectric focusing (IEF) applied in one direction, and gel electrophoresis (GE) applied at aright angle to the first separation direction (O Farrell, 1975 Celis and Bravo, 1984). In this case, proteins are first separated according to their isoelectric point, measured in p/units, and then according to their molecular weight by gel electrophoresis. The size separation step is usually aided by addition of a surfactant, most typically sodium dodecyl sulfate (SDS), and the gel material is a polyacrylamide formulation. 

Figure 8.9. Resolution of proteins from heart muscle by 2D electrophoresis isoelectric focusing (IEF) x gel electrophoresis (GE). Sample size, lmg wet weight silver stain. (Courtesy of E. Jellum, University of Oslo.)...

Gel electrophoresis (GE) is a common separation technique in protein analysis and it has also been used for the speciation of metals bound to proteins [86]. In most applications, metals have been detected by autoradiography, limiting the studies to those elements for which a relatively stable radionuclide exists [87]. As an example, 75Se radiotracer allowed Se to be detected after two-dimensional GE (2-DE) separation [88]. Owing to the high sensitivity and isotopic capability of ICP-MS, this technique has been proposed as the detection tool of choice for elements in gel. The efbcient transport of the sample from the protein spot on gel to plasma has been achieved by laser ablation (LA) [89, 90] and electrothermal (ET) atomization [62, 91] techniques. The... 

As discussed in Section 14.2.1, normal (one-photon) fluorescence spectroscopy (IPE) has been the method of choice in capillary electrophoresis (CE) and gel electrophoresis (GE) with visible and NIR fluorescent dyes. MPE fluorescence, however, is also quite suitable for detection in CE and GE. For instance. Song et al. fractionated coumarine dyes with capillary electrophoresis, and detected the dyes at at-tomole concentrations by 2PE fluorescence [189]. Generally, detection Hmits are comparable to those attainable by normal fluorescence. However, MPE is particu-... 

Both gel electrophoresis (GE) and CE have an extremely broad application potential for analysis, preparation, and characterization of proteins. 

Gel electrophoresis (GE) was developed in the 1940s, while capillary electrophoresis appeared 40 years later. Then chromatography with electric potential-driven liquid flow also developed into micellar electroldnetic chromatography (MEKC) and electrochromatography (EC), both with capillary columns. Electrophoresis, thus, is not a chromatographic technique, since there is no stationary phase, except in MEKC and EC. 

Hyphenated techniques are the analytical methods most frequently used today for elemental speciation. For that purpose, a separation technique, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), or gel electrophoresis (GE), is coupled on-line with an elemental detection method, such as ICP-MS [67]. Hyphenated ICP-MS techniques can also be combined with the isotope dilution method for quantification of elemental species. Two different spiking modes are possible, the species-specific and spedes-unspedfic mode. Rottmann and Heumann were the first to present a setup for H PLC-ICP-IDMS enabling the use of these two spiking modes [68] (Figure 8.14). 

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. 

Flatbed gel electrophoresis (GE) is used extensively in the analysis and characterization of proteins with a variety of protocols such as isoelectric focusing (lEF) and polyacrylamide gel electrophoresis (PAGE), but has not been specifically developed for the separation of metal-containing proteins. Therefore, before using the method for speciation of biological samples, some important parameters have to be checked. It has to be investigated in detail if speciation data are hampered by contamination or alteration of native species due to the influence of the electric field and/or denaturing buffer systems. ... 

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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