Electrophoretic gel method

Hirschfeldt (H8) has suggested that the small differences in migration rate of the different HpHb complexes in agar gel at pH 8.6 with a Ca++-containing barbiturate buffer can be used for ascertaining the Hp type. In our opinion the results obtained by this simplified procedure are less reliable than those obtained by the somewhat more complicated starch gel electrophoretic method. 

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). 

During the last decades the different modes of capillary electrophoresis (CE) have developed into high-performance (HPCE) separation methods, offering a high separation efficiency (lO -lO theoretical plates), high sensitivity (femtomole to zeptomole amounts in nano- to picoliter sample volumes), and short analysis times (typically 5-20 min, in special cases only a few seconds). They are considered as a recognized complement and/or coimterpart of liquid chromatography (LC) and gel electrophoretic methods. 

Another possibility is to use a capillary gel electrophoretic method that is nowadays a routinely and commercially available method for the determination of the molecular mass of proteins/polypeptides. This method can also be used for the separation of collagen chains and their polymers. For example, this procedure is described in the literamre for the separation of collagen type I a-chains and chain polymers p (dimers), and y (trimers), and also chain polymers of related molecular mass 300,(X)0 and higher (typically in the study of the formation of crosslinks). Besides commercially available kits, another option is to use fused-silica or poly vinylalcohol-coated capillaries filled with non-cross-linked polyacrylamide or hydroxyl-propylmethylcellulose in a 50 vaM Tris-glycine buffer (pH 8.8) or phosphate buffer (50 vaM, pH 2.5) (Table 1). 

To date, classical gel electrophoretic methods have been well established and are frequently used in research, especially in molecular biology and thus, in clinical practice. On the other hand, electrophoretic methods performed in capillaries without any supporting media are making great progress and hold the promise of extensive application in the near future. 

The present development of capillary electrophoresis as a separation method in a free solution substituting the classical gel electrophoretic methods, is undergoing great progress. Over the last few years, many new reports have been devoted to a better understanding of the separation processes during electrophoresis in capillaries [68-74]. There is constantly increasing interest in the application and the successful separation of various substances [75-78] by capillary electrophoresis. 

The SDS-acrylamide gel electrophoretic method used by Hoober and coworkers separates the proteins designed by Eytan and Ohad as L protein(s) into three fractions polypeptides a, b, and c. Modifications of the SDS-gel electrophoresis method according to Laemli yield still another pattern in which the L protein(s) are separated into four bands (III, IV, Va, Vb). " ... 

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. AnalyMcal uses of this technique center around protein characterization, for example, purity, size, or molecular weight, and composition of a protein. Polyacrylamide gels can be used in both reduced and nonreduced systems as weU as in combination with discontinuous and ief systems (39). 

In the previously described electrophoretic methods, the capillary was filled with electrolytes only. Another mode of operation in capillary electrophoresis involves filling the capillary with gel or viscous polymer solutions. If desired, a column can be packed with particles and equipped with a frit.68 This mode of analysis has been favorably used for the size determination of biologically important polymers, such as DNA, proteins, and polysaccharides. The most frequently used polymers in capillary gel electrophoresis are cross-linked or linear polyacrylamide,69 cellulose derivatives,70-75 agarose,76 78 and polyethylene glycols. 

O Farrell PH et al. Two-dimensional polyacrylamide gel electrophoretic fractionation. Methods Cell Biol 1977 16 407-420. 

Over the past few years, a large number of experimental approaches have been successfully used as routes to synthesize nanorods or nanowires based on titania, such as combining sol-gel processing with electrophoretic deposition,152 spin-on process,153 sol-gel template method,154-157 metalorganic chemical vapor deposition,158-159 anodic oxidative hydrolysis,160 sonochemical synthesis,161 inverse microemulsion method,162 molten salt-assisted and pyrolysis routes163 and hydrothermal synthesis.163-171 We will discuss more in detail the latter preparation, because the advantage of this technique is that nanorods can be obtained in relatively large amounts. 

As with other chromatographic methods, there are a number of electrophoretic methods, including paper and gel electrophoreses and CE. Electrophoresis uses an electric current to move ionic species, either simple ions, amino acids, or complex proteins, through a medium (i.e., a gel) or a capillary (i.e., CE). During this process, typically, the ionic species move at different rates and are thus separated. 

Gel electrophoresis is widely used in the routine analysis and separation of many well-known biopolymers such as proteins or nucleic acids. Little has been reported concerning the use of this methodology for the analysis of synthetic polymers, undoubtedly since in many cases these polymers are not soluble in aqueous solution - a medium normally used for electrophoresis. Even for those water-soluble synthetic polymers, the broad molecular weight dispersities usually associated with traditional polymers generally preclude the use of electrophoretic methods. Dendrimers, however, especially those constructed using semi-controlled or controlled structure synthesis (Chapters 8 and 9), possess narrow molecular weight distribution and those that are sufficiently water solubile, usually are ideal analytes for electrophoretic methods. More specifically, poly(amidoamine) (PAMAM) and related dendrimers have been proven amendable to gel electrophoresis, as will be discussed in this chapter. 

Electrophoretic Methods Systems for polyacrylamide gel electrophoresis, 104, 237 preparative isoelectric focusing,104, 256 gel... 

Electromigration methods compose a family of analytical separation methods based on differences in the mobilities of charged analytes in the electric field. In this chapter, we discuss mainly such electromigration methods that are performed in thin capillaries with inner diameter (i.d.) <0.1 mm. These methods are commonly known as capillary electrophoretic methods where the most important modes are capillary zone electrophoresis (CZE), micellar electrokinetic capillary chromatography (MEKC), capillary gel electrophoresis (CGE), and capillary electrochromatography (CEC). 

Electrophoretic Methods. Several electrophoretic procedures have been developed to fractionate or purify the various caseins (McKenzie 1971C Thompson 1971 Whitney 1977). Wake and Baldwin (1961) fractionated whole casein by zone electrophoresis on cellulose powder in 7 M urea and 0.02 ionic strength sodium phosphate buffer at pH 7 and 5°C. Payens and co-workers employed several somewhat different electrophoretic conditions for the fractionation and purification of the caseins on cellulose columns (Payens 1961 Schmidt and Payens 1963 Schmidt 1967). Three fractions, as-, k-, and /3-caseins, were separated at pH 7.5 and 30°C with 4.6 M urea-barbiturate buffer. The purification of asi-casein and the separation of the genetic variants of K-casein were accomplished by altering the electrophoretic conditions. Manson (1965) fractionated acid casein on a starch gel column stabilized by a density gradient at 25 °C. 

The use of agarose as an electrophoretic method is widespread. The advantages uf agarose electrophoresis are that it requires no additives of cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Techniques have also been developed for the specific visualization of particular classes of enzymes following electrophoretic separation in a gel. These techniques are often referred to as activity staining, as the intrinsic activity of the enzyme is used, either to produce a colored product or to produce a clear zone on a colored background within the gel. A method for visualizing proteinases based on the work of Gar-cfa-Carreno and Haard (1993) and Garcia-Car-reno et al. (1993) is presented (see Basic Protocol 3). 

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