Electrophoresis

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Electrophoresis is the process in which macromolecules (such as proteins, DNA, or RNA fragments) are charged and move in response to an electric field. Electrophoretic mobility is a result of a constant drift speed, s, reached by an ion when the driving force zeE (where ze is the net charge and E is the field strength) is matched by the frictional force fs. The drift speed is then:

s=\frac{zeE}{f}

Therefore, the mobility of a macromolecule is an electric field depends on its net charge, size (and hence molar mass), and shape. The latter two factors are implied by the dependence of s on f.

The drift speeds attained by polymers in traditional electrophoresis methods are rather low; as a result, several hours are often necessary to effect good separation of complex mixtures. According to the drift speed equation above, one way to increase the drift spreed is to increase the electric field strength. However, there are limits to this strategy because very large electric fields can heat the large surfaces of an electrophoresis apparatus unevenly, leading to a non-uniform distrobution of electrophoretic mobilities and poor separation.

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Capillary Electrophoresis

In capillary electrophoresis, the sample is dispersed in a medium (such as methyl-cellulose) and held in a thin glass or plastic tube with diameters ranging from 20 to 100 um. The small size of the apparatus makes it easy to dissipate heat when large electric fields are applied. Excellent separations may be effected in minutes rather than hours. Each polymer fraction emerging from the capillary can be characterized further by other techniques, such as Matrix-assisted laser desorption/ionization - time of flight analyser or MALDI-TOF.

Gel Electrophoresis

An important tool in genomics and proteomics is gel electrophoresis, in which biopolymers are sparated on a slab of a porous gel, a semirigid dispersion of a solid in a liquid. Because the molecules must pass through the pores in the gel, the larger the macromolecule the less mobile it is in the electric field and, conversely, the smaller the macromolecule the more swiftly it moves through the pores. In this way, gel electrophoresis allows for the separation of components of a mixture according to their molar masses.

Agarose Gel Electrophoresis

Agarose is gelatinous substance derived from seaweed. The gelling agent is an unbranched polysaccharide obtained from the cell membranes of some species of red algae. Chemically, it is a polymer made up of subunits of the sugar galactose. Agarose polysaccharides serve as the primary structural support for the algae's cell walls. The neutral charge and lower degree of chemical complexity of agarose make it less likely to interact with biomolecules, such as proteins. Gels made from purified agarose have a relatively large pore size, making them useful for size-separation of large molecules, such as proteins or protein complexes >200 kilodaltons, or DNA fragments >100 basepairs thus, better suited for the study of large macromolecules, such as DNA and enzyme complexes.

Polyacrylamide Gel Electrophoresis

Two-dimensional (2D) Electrophoresis

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