Electrophoresis support material

Immunoaffinity procedures have also been developed to selectively extract corticosteroids from different sample matrices. Thus, Seymour et al. demonstrated the higher efficiency of the immunoaffinity methods compared with the conventional extraction procedures using organic solvents [177]. Immunosorbents have also been used for online procedures followed by HLPC-UV [178, 179], HPLC-APCI-MS [179,180], GC-MS [176,181], or capillary electrophoresis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. The online IAC-HPLC-MS allowed determination of dexamethasone and flumethasone in equine urine with LODs in the range 3-4 ng mL-1 [180]. The cross-reactivity values obtained in the ELISA and the recoveries of an IAC-HPLC procedure are presented in Table 7. Bagnati et al. developed an immunoaffinity extraction... 

Zone electrophoresis is normally carried out horizontally in a suitable medium such as paper, polyacrylamide gel, starch gel or cellulose acetate. The sample components can be completely separated and quantitatively and qualitatively identified in much lower quantities than by the moving-boundary method. The procedure consists of saturating the support material with a buffer solution. The ends of the strip of support are immersed in separate reservoirs of buffer solution to maintain the saturation. The sample is then applied as a narrow band near one end of the support strip. A voltage potential is created down the length of the strip causing the sample components to ionize and then migrate at a rate dependent on their charge, molecular size and interactions with the support medium. When the process is complete, the strip is removed and developed for examination of the separated components. Densitometry is normally used for quantitation of the bands after suitable color development. 

The unidimensional type of paper electrophoresis is an extension of free boundary electrophoresis, the method developed by Tiselius (Tl). There are several differences between the two systems. One is the presence of a substrate (supporting medium) as anticonvectional medium during the electrophoretic separation. Another important difference is the starting point. In paper electrophoresis the entire load of material due to be separated is collected on the starting line, whereas in free boundary electrophoresis the material is present in equal concentration over one leg of the electrophoretic cell. Fortunately these differences simplify the qualitative and quantitative appraisal of separation after the run on paper, and for practical work both prove to be true inherent qualities and go far to account for the success of the method (Kl, VI, Wl). 

Electrophoresis in a free electrolyte without a support material is most straightforward and avoids complicating interactions with the support, but it is clearly convection prone. 

Electrophoretic Separation. The net charge on a particular protein varies with the pH of the medium in which it is dissolved. Accordingly, application of an electric field to a buffered, heterogeneous protein solution often results in their differential migration in free solution or in heterogeneous systems with an inert supporting material, such as a polyacrylamide gel. Because of the difficulty of scaling up electrophoretic procedures, they are more commonly used for analytical applications than for the preparative scale purification of proteins. The use of electrophoresis is discussed in more detail in Experiment 4. 

Fluorous reverse phase silica gel (FRPSG) has been used in the purification of synthetic DNA fragments.In solid phase DNA synthesis, truncated sequences are often separated from the desired product after deprotection using HPLC or electrophoresis. In order to perform, parallel syntheses and separations of nucleotides the trityl-on purification procedure was developed, in which a lipophilic support material is used to separate the desired and undesired product, followed by deprotection. If the protecting group is labelled with a fluorous group, fiuorous-fiuorous interactions between the FRPSG and the protected nucleotide can be used to aid separation of the aqueous mixture. 

A number of apparatuses were constructed for this purpose [257-264], some of which are commercially available. Here we describe the apparatus marketed by LKB (Bromma, Sweden) (see also Ref. 264) (Fig. 6.26). The apparatus consists of a inverted U-shaped tube that is composed of two parts connected by a joint in the upper part. One limb accommodates the electrophoresis column that is surrounded by a covering jacket, the other limb filled with buffer serves to make connection with the electrode vessel. At the lower end of the column a collecting funnel is attached. Liquid connection or disconnection between the two limbs can be done through a ground female joint. The stopcock above this joint connects the system to a buffer reservoir when the column is washed or when the zones are eluted. In the male joint of the top piece there is a hole that allows liquid to pass to the other limb. By turning the liquid stream can be disconnected at this point. The column size is 55 X 2 cm Sephadex or cellulose serve as the supporting material. Separation is run at 500-1000 V and 25-50 mA. 

In zone electrophoresis a homogeneous solid or gel framework is used to support the solution and minimize convectional effects arising from temperature gradients due to Joule heating. In this procedure the supporting material is saturated with the buffer solution, and a small amount of the macromolecular... 

In comparison with common zone electrophoresis and with chromatographic techniques, capillary isotachophoresis does not employ any supporting material so that permanent stability and reproducibility of the working conditions can easily be secured. It is only slightly affected by accompanying non-ionic substances that often cause deterioration of chromatographic columns. In isotachophoresis, non-ionic accompanying compounds remain at the injection part and, are washed out from the separation capillary after analysis has been performed. 

Alicea-Maldonado, R. and Colon, L., Capillary electrochromatography using a fluoropolymer as the chromatographic support material. Electrophoresis, 20, 37, 1999. 

Free-flow electrophoresis (FFE) is a continuous run operation, without the need for supporting materials, and an interesting alternative to preparative chromatographic techniques. Therefore, FFE is a labor-saving method and well suited for purification of labile biopolymers, since in contrast to chromatography, there are no disturbing interactions with stationary phases that often induce a loss in biological activity. 

A technique that is used for the identification of proteins. It consists initially of separation of the proteins of the serum, or other fluid, by electrophoresis. This is usually carried out in agarose, although other support materials can be used. After electrophoresis, antiserum is placed in a trough adjacent and parallel to the direction of electrophoresis and the proteins and antibodies are allowed to diffuse towards each other. Interaction between the antibodies and the protein antigen results in the precipitation of immune complexes within the gel, which takes the form of an arc or bow for each protein. Thus if human serum has been separated and polyvalent antiserum has been placed in the trough, a series of arcs is obtained, each arc resulting from the precipitation of a serum protein with its own specific antibody. Polyvalent antiserum or monospecific antisera (anti-IgG, anti-IgA etc.) can be placed in the troughs. In the case of mono-specific antiserum, usually only one precipitin bow results. 

Electrophoresis fraction No. 4 was chosen for further chemical characterization, in spite of the fact that it was not as antioxidative as the precipitate. It was, however, considered to be more homogeneous. At least it was purified from the fluorescent material. Still, its purity is uncertain. The fact that no well defined bands were obtained in the electrophoresis indicates that the fractions consisted of more than one compound, possibly differing slightly in molecular weight and/or content of charged groups. Another explanation to the lack of distinct bands could be unselective adsorption between the compound and the paper. This is supported by the fact that rechromatography of the five electrophoresis fractions failed to reproduce the same fractions distinctly. 

Zone electrophoresis is used mainly as an analytical technique and, to a lesser extent, for small-scale preparative separations. The main applications are in the biochemical and clinical fields, particularly in the study of protein mixtures. Like chromatography, zone electrophoresis is mainly a practical subject, and the most important advances have involved improvements in experimental technique and the introduction and development of a range of suitable supporting media. Much of the earlier work involved the use of filter paper as the supporting medium however, in recent years filter paper has been somewhat superseded by other materials, such as cellulose acetate, starch gel and polyacrylamide gel, which permit sharper separations. 

In the separation of proteins by electrophoresis, the sample is submitted to an electrical field, causing the electrically charged proteins to move in the direction of the applied current. If the experiment is carried out in solution (free electrophoresis) and the proteins have different charge densities, they will move with different velocities, allowing their separation. In practice, the process is normally carried out in a gel matrix, instead of in solution, and the gel can act not only as an inert support for the electrophoresis buffer, but also, if desired, as an active material that interacts with the proteins. 

Most practical applications of electrophoresis in biochemistry employ some form of zonal electrophoresis, in which the aqueous ionic solution is carried in a solid support and samples are applied as spots or bands of material. Paper electrophoresis, cellulose acetate strip and cellulose nitrate strip, and gel electrophoresis are all examples of zonal... 

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