Electrophoresis preparative techniques

Pridmore, A.M., Devine, D.A., Bonass, W.A. and Silley, P. (1999) Influence of sample preparation technique on two-dimensional gel electrophoresis of proteins from Porphyromonas gingivalis. Letters in Applied Microbiology 28, 245-249. 

Electrophoresis plays a key role as an analytical or preparative technique in the characterization of natural organic matter because it gives information about the behavior of these molecular mixtures in controlled solution conditions, depending on both the size and the charge distribution frequency of the analytes in the complex mixture. Historically, the first electrophoretic separations were conducted with environmental colloids and over the years all the techniques based on zone, gel electrophoresis, or isoelectric focusing were used in their different setups to analyze natural organic matter and environmental particles to a minor extent. The goal of... 

Using a single capillary to collect a separated component may present a problem to the user (from the point of view of quantity). Currently, capillary electrophoresis is used primarily for analytical tests. However, two approaches have been performed to use capillary electrophoresis as a micro- or semi-preparative technique. One approach is done by increasing sample load and detector response by arranging capillaries in bundles (85). The ideal instrument should be configured to... 

Electrophoretic techniques have a number of practical advantages, such as high resolution, equipment of relative simplicity and low cost, feasibility for multiple samples, high sensitivity, easy detection of specific molecules, and availability of standards giving reasonably well defined bands. However, as a consequence of its low capacity, electrophoresis is mostly used for analytical purposes rather than as a preparative technique. More details on electrophoretic techniques are provided in Chapter 13. 

As indicated above, electrophoresis represents an important method utilizing an electrical field to move charged particles or molecules with different physical properties through a medium. This section will focus on the set up and use of the basic equipment for analytical gel electrophoresis, which may serve as a precursor (preparative technique) to additional analytical methods including mass spectrometry, PCR and chromatography. 

In general, the resolution is inferior to acrylamide gel electrophoresis and zone centrifugation but in situations where high resolution is not required gel filtration is a useful, reliable and predictable preparative technique. 

Similarly, the sample preparation must be complementary to the instrumental method. Chen and Pollack (1) compared four sample preparation techniques for a capillary electrophoresis assay of a peptide in human plasma. Table 3 is a... 

An alternative electrochemical source separation and preparation technique is electrophoresis. A drop of the radionuclide solution is pipetted at one end of a moistened paper strip that has an electric potential difference along its length. Each ionic radionuclide moves at its own rate down the strip. Locations of individual radionuclides are identified by their radiation or by dyes affected by the chemical form (Deyl 1979). 

As the zone separation resolves the sample components in a much better manner, its use as a preparative technique is logical. The free electrophoresis on the other hand cannot be modified for the purpose, as it is difficult to isolate Individual components of a sample by this method. 

Liquid-phase microextraction (LPME) as a sample preparation technique for chromatography and electrophoresis is one of the recently developing techniques. In LPME, the principles of LLE and the miniaturized nature of SPME are combined to realize the advantages of both techniques. 

Paper Electrophoresis. Paper (qv) as an electrophoretic matrix was employed in some of the first electrophoretic techniques developed to separate compounds. Paper is easier than a gel matrix because the paper matrix requires no preparation. Besides being easy to obtain, paper is a good medium because it does not contain many of the charges that interfere with the separation of different compounds. Two types of paper employed in this type of electrophoresis are Whatman 3 MM (0.3 mm) and Whatman No. 1 (0.17 mm). 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

A number of developments have increased the importance of capillary electrophoretic methods relative to pumped column methods in analysis. Interactions of analytes with the capillary wall are better understood, inspiring the development of means to minimize wall effects. Capillary electrophoresis (CE) has been standardized to the point of being useful as a routine technique. Incremental improvements in column coating techniques, buffer preparation, and injection techniques, combined with substantive advances in miniaturization and detection have potentiated rugged operation and high capacity massive parallelism in analysis. 

The beauty of 2D gel electrophoresis as a separation technique is the orthogonality of the two separation dimensions separation by charge (isoelectric point) in the first dimension, and separation by size in the second dimension. Two-dimensional gel electropheresis is the core separation technique for proteomics, along with HPLC (for preparative isolation). 

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