Electrophoretic Support Media

Figure 2 Cross-sectional view of an electrophoretic apparatus. A, Furnace B, electrophoresis chamber C, capillary D, tube supporting the capillary E, screw to adjust the height of the tube F, electrophoretic support medium G, heat-resistant glass plate H and H graphite electrodes I and I, reservoirs L, thermal couple M, heating wire, and N, insulating jacket. (Reproduced with permission from Alberti G, Allulli A, and Modugno G (1964) Journal of Chromatography 5 420-429.)...

A material which can be used as an electrophoretic support medium. It is supplied commercially as membranes, which contain a large proportion of air spaces. These air spaces become filled with liquid when wetted, transforming the membrane into a pliable sheet. One of the major advantages of cellulose acetate as an electrophoretic support medium is that adsorption is minimal, enabling sharply defined bands to be obtained. The sheets can be made transparent for densitometric measurements by the use of suitable solvents. 

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. 

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). 

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. 

On the other hand, the intervening media used in electrophoresis have much lower conductivity, and an equivalent circuit for an electrophoresis cell includes a resistor between the capacitors at the electrode-solution interfaces. Across the support medium, potential is now (usually) a linear function of distance, and the electric field thus generated is responsible for driving the electrophoretic separation. Electrophoresis occurs at the electrodes used in electrophoresis, to maintain the... 

To compensate for experimental variations in E, the electrophoretic mobility of a particle has been defined as a fundamental characteristic of a molecule under given conditions of support medium, pH, ionic strength, and temperature. The electrophoretic mobility, p, usually reported in units of velocity per unit field strength, is given by Eq. 9.3 ... 

Another factor and potential problem that can affect mobility is wick flow. During electrophoresis heat evolved because of the passage of current through a resistive medium can cause evaporation of solvent from the electrophoretic support. This drying effect draws buffer into the support fi om both buffer compartments. If significant, the flow of buffer from both directions can affect protein migration and hence the calculated mobility. 

Proteins are charged at a pH other than their isoelectric point (pi) and thus will migrate in an electric field in a manner dependent on their charge density. If the sample is initially present as a narrow zone, proteins of different mobilities will travel as discrete zones and thus separate during electrophoresis. Such separations are best carried out in a support medium to counteract the effects of convection and diffusion during electrophoresis and to facilitate immobilization of the separated proteins. Polyacrylamide gel, the use of which dates back to 1959 (1-3), has proved to be a versatile and popular matrix for the electrophoretic separation of proteins. 

An electrophoretic separation is performed by injecting a small band of the sample into an aqueous butler solution contained in a narrow tube or on a flat porous support medium such as paper or a semisolid gel. A high voltage is applied across the lenulh of the buffer by means of a pair of electrodes located at each... 

Since suspended or colloidal particles usually bear an electric charge which is compensated only by a distributed atmosphere of counterions (gegenions) out in the support medium, the electrophoretic motion of the charges due to an applied field could give a dielectric response. It turns out upon detailed analysis that the response is negative in that the contribution is such as to decrease the overall polarization of the system. Since this has not been observed to date in biological systems, we drop further discussion of it here. 

The supporting medium where electrophoresis takes place is called the electrophoretic system or the separation system. The separation systems are solutions of electrolytes that are filled into a separation column or a separation charmel (Figure 1). Both ends of the column are connected with vessels, which are also filled with the solution of electrolytes and serve as a stock of the solution. The electric voltage (500-30000 V), which is called driving voltage, is provided by two electrodes in the two vessels and... 

It is evident from the foregoing discussion that an exact determination of the electrophoretic mobility, which can be utilised for identifying a substance, is accompanied in heterogeneous systems by many difficulties. In electrophoresis with supporting medium, one thus works in practice with the concept of apparent instead of real mobility. 

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