Electrophoresis supporting media

There is often a need for a visualization procedure that is specific for a certain biomolecule, for example, an enzyme. If the enzyme remains in an active form while in the gel, any substrate that produces a colored product could be used to locate the enzyme on the gel. Although it is less desirable for detection, the electrophoresis support medium may be cut into small segments and each part extracted with buffer and analyzed for the presence of the desired component. 

Capillary Electrophoresis. Capillaries were first appHed as a support medium for electrophoresis in the early 1980s (44,45). The glass capillaries used are typically 20 to 200 p.m in diameter (46), may be filled with buffer or gel, and are frequendy coated on the inside. Capillaries are used because of the high surface-to-volume ratio which allows high voltages without heating effects. The only limitations associated with capillaries are limits of detection and clearance of sample components. 

Raymond, S Weintranb, LS, Acrylamide Gel as a Supporting Medium for Zone Electrophoresis, Science 130, 7111,1959. 

Raymond, S. and Weintraub, L., Acrylamide as a supporting medium for zone electrophoresis, Science, 130, 711, 1959. 

Factors Affecting Ionic Migration. Effect of Temperature. pH and Ionic Strength. Electro-osmosis. Supporting Medium. Detection of Separated Components. Applications of Traditional Zone Electrophoresis. High-performance Capillary Electrophoresis. Capillary Electrochromatography. Applications of Capillary El ectrochromatography. ... 

Traditional electrophoresis paper, cellulose acetate or polymeric gels used as a supporting medium for the electrolyte solution enclosed tank with electrodes and buffer reservoirs dc power supply. 

Traditional electrophoresis involves the differential migration of charged species in an electrolyte solution under the influence of an applied potential gradient. The rate of migration of each species is a function of its charge, shape and size. In traditional zone electrophoresis, the electrolyte solution is retained by an inert porous supporting medium, usually paper or gel, in the form of a sheet or column. Application of a dc potential across the solution for a period of time results in the components of a mixture, originally placed... 

Electrophoresis can be carried out using paper or a gel as the supporting medium. Typically, it can only be carried out in media compatible with water because buffers or salt solutions are required to carry the electric current required for separation. CE is carried out in a fused-silica capillary filled with buffer. 

Polyacrylamide gel is the most commonly used type of support medium for gel electrophoresis, and polyacrylamide gel electrophoresis is simply known as PAGE. The gel is usually formed by polymerization of acrylamide and the cross-linking agent N, iV -methylene-bis-acrylamide (Bis) in the presence of ammonium persulfate (APS, initiator) and N, N, N, iV -tetramethyl-ethylenediamine (TEMED, accelerator). The total concentration of acrylamide... 

Zonal techniques are the most frequently used form of electrophoresis and involve the application of a sample as a small zone to a relatively large area of inert supporting medium which enables the subsequent detection of the separated sample zones. A wide range of supporting media have been developed either to eliminate difficulties caused by some media (e.g. the adsorptive effects of paper) or to offer additional features (e.g. the molecular sieving effects of polyacrylamide gel). 

HDL, respectively. By starch block, some of the VLDL exhibit 0 2-mobility, whereas by polyacrylamide gel electrophoresis the pre-j8 band migrates in post-)8 position, a phenomenon due to the sieving effect of the supporting medium. 

In 1956, Smithies and Poulik first used 2-DE combining paper and starch gel electrophoresis to separate serum proteins. Nearly 20 years later, polyacrylamide was applied as a support medium. Charge-based protein separation followed as isoelectric focusing (IEF), applied to SDS-PAGE. Later, urea and nonionic detergents were used in IEF-2DE. The most significant achievement was the separation of proteins from E. coli. 

In theory, if the net charge, q, on a molecule is known, it should be possible to measure / and obtain information about the hydrodynamic size and shape of that molecule by investigating its mobility in an electric field. Attempts to define /by electrophoresis have not been successful, primarily because Equation 4.3 does not adequately describe the electrophoretic process. Important factors that are not accounted for in the equation are interaction of migrating molecules with the support medium and shielding of the molecules by buffer ions. This means that electrophoresis is not useful for describing specific details about the shape of a molecule. Instead, it has been applied to the analysis of purity and size of macromolecules. Each molecule in a mixture is expected to have a unique charge and size, and its mobility in an electric field will therefore be unique. This expectation forms the basis for analysis and separation by all electrophoretic methods. The technique is especially useful for the analysis of amino acids, peptides, proteins, nucleotides, nucleic acids, and other charged molecules. 

The electrophoretic techniques discussed up to this point are useful for analyzing proteins and small fragments of nucleic acids up to 350,000 daltons (500 bp) in molecular size however, the small pore sizes in the gel are not appropriate for analysis of large nucleic acid fragments or intact DNA molecules. The standard method used to characterize RNA and DNA in the range 200 to 50,000 base pairs (50 kilobases) is electrophoresis with agarose as the support medium. 

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. 

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. 

Initially, electrophoresis was performed in gel or other media in the form of a bed, slab, rod, etc., but due to laborious multistage handling of supporting media and nonreproducibility of the results the supporting medium was replaced by a capillary and the technique was called capillary electrophoresis (CE). More recently, the silica capillary was replaced by a microchip and the technique is called microchip electrophoresis we define it as nanocapillary electrophoresis (NCE) as it deals with all aspects at nano or low levels of... 

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). 

In two-dimensional electrophoresis the charged particle migrates in a field of two forces which act perpendicularly to one another. The first force F creates a vertical hydrodynamic field. A flow of liquid runs by gravity down a vertical curtainlike supporting medium to which we shall refer as the substrate. The liquid is a buffer solution which through its pH and ionic strength determines the mobility of the particle. 

To reduce adsorption of cellulose powder (T2) Flodin introduced ethanol-treated cellulose as a supporting medium for zone electrophoresis columns (FI). The main advantage is low adsorption, so that the column can be eluted and used over and over again. In the large models up to 2 liters of serum are separated, while microcolumns are under development which should give excellent results for clinical work (PI, Tl). 

The electrophoretic separation technique is based on the principle that, under the influence of an applied potential field, different species in solution will migrate at different velocities from one another. When an external electric field is applied to a solution of charged species, each ion moves toward the electrode of opposite charge. The velocities of the migrating species depend not only on the electric field, but also on the shapes of the species and their environmment. Historically, electrophoresis has been performed on a support medium such as a semisolid slab gel or in nongel support media such as paper or cellulose acetate. The support media provide the physical support and mechanical stability for the fluidic buffer system. Capillary electrophoresis (CE) has emerged as an alternative form of electrophoresis, where the capillary wall provides the mechanical stability for the carrier electrolyte. Capillary electrophoresis is the collective term which incorporates all of the electrophoretic modes that are performed within a capillary. 

The reduction of the thickness of the flow channel, as discussed earlier, is equivalent to introducing more surface area per unit volume of medium. High surface areas inhibit all flow, including natural convective flow. One can increase relative surface areas by going to thinner tubes or channels, or by using a fine granular or porous support medium. Both approaches are used in electrophoresis as discussed in a subsequent chapter. 

---