Thin-layer slab gels

Detection of proteins on thin-layer plates, gel slabs, or membranes is often accomplished by staining with a dye,265-267 the most widely used being Coomassie brilliant blue.268 Various silver-containing stains may also be used. After separation of a protein mixture by electrophoresis and transfer to an inert membrane,... 

The thin-layer gel system used for resolving the different fragments produced by the chemical degradation method is essentially that of Sanger, Nicklen and Coulson (1977) as described in Chapter 4. An 8% polyacrylamide gel is usually used though for special purposes a 10 or 12% gel can give an improved resolution of the smallest fragments. For the standard 8% gel the polymerization mixture contains 7.6% (wt/vol) acrylamide, 0.4% (wt/vol) bisacrylamide, 50% (wt/vol) urea, 100 mM Tris-borate, pH 8.3, 2mM EDTA, 0.07% (wt/vol) ammonium persulphate and TEMED catalyst. This solution is poured or injected into a 0.4 x 200 x 400 mm mold to form a gel slab. 

Ultra-thin-layer gel electrophoresis is a combination of slab-gel electrophoresis and capillary gel electrophoresis [130], providing a multilane separation plat-... 

Forensic science ink analysis is one of the most traditional analytical fields. Currently, thin-layer and column chromatography as well as slab gel electrophoresis are used to investigate ink composition, but, recently, CZE has been tentatively applied, with encouraging results (Fanali and Schudel, 1991). 

Electrophoresis has been used as a separation technique for decades, particularly by biochemists, in the open-bed format. In this mode, a layer of a gel is formed on a flat-bed support which is in contact with an electrolyte and two electrodes are situated at either end of the open slab. The sample is placed at one end of the separation medium, and when voltage is applied, the molecules migrate through the gel by electrophoresis. The components in the sample are separated based on their differences in electrophoretic mobility. The electrophoretic mobility is controlled by molecular parameters such as charge, size, and shape. After the electric field is turned off, the separation is evaluated by spraying the plate with a dye and the bands of the sample components become visible, similar to the detection format used in paper or thin-layer chromatography. 

In conclusion, CE is a valuable analytical tool that offers a number of possibilities for the analysis of a wide spectrum of forensicaUy interesting compounds. Practically all compounds which have been traditionally analyzed by GC, high-performance Uquid chromatography, thin-layer chromatography, or slab-gel electrophoresis, can be assayed by capillary electrophoretic procedures. AU methods of capillary electrophoresis can be validated and can meet the demands of good laboratory practice. 

In conclusion of this section it is necessary to mention the possibility to elute several polyacrylamide gel slabs or rods that are segmented, before elution, according to a marker gel (the method is analogous to preparative thin-layer or paper chromatography). More information in this respect can be gained from Refs. 

Electrophoretic separations are currently performed in two quite different formats one is called slab electrophoresis and the other capillary electrophoresis. The first is the classical method ihal has been used for many years to separate complex, high-molecular-mass species of biological and biochemical interest. Slab separations arc carried out on a thin flat layer or slab of a porous semisolid gel containing an aqueous buffer solution within its pores. This slab has dimensions of a few centimeters on a side and, like a chromatographic thin-layer plate, is capable of separating several samples simultaneously. Samples arc introduced as spots or bands on the slab, and a dc electric held is applied across the slab for a fixed period. When the separations are complete, the Held is discontinued and the separated species are visualized by staining in much the same way as was described for thin-layer chromatography in Section 281-2,... 

Prior to the development of CZE, the primary application of electrophoresis was for large biological macromolecule separations on planar gels. The gels were supported on glass plates not unlike the particles in the thin layer of a TLC plate. These so-called slab gels were needed to confine the separation buffer. It was easier to prepare cross-linked... 

The final compounds are then isolated by any of the fast selective procedures available. These include thin-layer chromatography (tic) on polyethyleneimine (PEI) cellulose plates, slab preparative gel electrophoresis, and hplc on Per-maphase AAX or Partisil lOSax column. 

Hitherto, gel electrofocusing has been carried out in thin layers and troughs, or else in narrow tubes. Very thin layers have been used by Awdeh et cU. (39), as well as by Catsimpoolas (28). Leaback and Rutter (37) used a special trough for casting slabs of polyacrylamide. The latter method permits a certain amount of preparative work. Riley and Coleman (30) also used thin layers. 

ITP as an experimental method has found wide application in analytical separation methodology, and it is also possible to operate prepara-tively in a continuous flow mode. The separation is provided in capillary tubes, thin-layer equipment, or gel rods, slabs or columns. Its designation as an independent method relies entirely upon operational considerations because the underlying theory and the separation process are in fact identical with the process that occurs during the stacking phase of electrophoresis in multiphasic buffer systems. 

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