Electrophoresis combined methods

Capillary electrophoresis combines the separation principles of gel electrophoresis with the throughput and detection methods of HPLC. It overcomes the disadvantages of slab gel electrophoresis, including slow and labor intensive procedures and the difficulty and inaccuracies of quantitation. CE is... 

In order to obtain information about the polarographic properties of the individual protein components in blood sera, the polarography was combined with paper electrophoresis. After electrophoretic separation, the cut strips of paper with separated fractions of albumins and globulins are eluted in physiological sodium chloride solution and each sample is analyzed polarographically. These combined methods were applied for study of various pathological cases [147]. 

Via. Verschure, J. C. M., van der Schaar, C. W., and Hadders, G., Comparative studies of the protein fractions from human gastrointestinal juices with paper electrophoresis combined with various detection methods. Protides Biol. Fluids, Proc. CoUoq., Bruges, 1959 7, 194-201. Elsevier, Amsterdam (1960). 

Other equally important methods are the electrophoretic techniques, such as polyacrylamide gel electrophoresis, isoelectric focusing, Western blots, combined electrophoresis, and isoelectric focusing (two-dimensional electrophoresis). Bioactivity methods are other key methods used in biotechnology product development. These include in vivo whole animal bioassay, cell culture bioassay, immunoassay, and biochemical assay. Many references and several textbooks are available in many industrial and academic libraries to provide additional and up-to-date information. 

It is possible to combine different methods of electrophoresis to give much better resolution of complex mixtures of proteins. For example, it has been estimated that the number of different proteins in an individual cell may be between 5000 (bacteria) and 50,000 (human). Many of these will have similar size or charge or pi, and resolution of so many bands on a single gel would be impossible. 2-D electrophoresis combines the techniques of SDS-PAGE and isoelectric focusing to give improved separation (Figure 7.11). 

Park, S.G, et al. (2002) A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography. Proteomics, 2 (2), 206-211. 

Asami, T. Imura, H. Absolute determination method for trace quantities of enantiomer of glufosinate by 7-cyclodex-trin modified capillary zone electrophoresis combined with solid-phase extraction and on-capillary concentration. Anal. Sci. 2006, 22, 1489-1493. 

There are three distinct modes of electrophoresis zone electrophoresis, isoelectric focusing, and isotachophoresis. These three methods may be used alone or in combination to separate molecules on both an analytical (p.L of a mixture separated) and preparative (mL of a mixture separated) scale. Separations in these three modes are based on different physical properties of the molecules in the mixture, making at least three different analyses possible on the same mixture. 

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). 

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

Two-Dimensional Electrophoresis. Two-dimensional (2D) electrophoresis is unique, offering an analytical method that is both reproducible and sensitive. It is referred to as 2D because it employs two different methods of electrophoresis, in two different dimensions, to produce one result. Each method separates the sample compounds based on different properties of each compound. The combination of the two methods gives better resolution of the compounds in the sample than could be achieved with either method alone. For example, each method alone may separate up to 100 components of a sample, whereas together they may separate up to 10,000 components. 

---