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Gel Electrophoresis - Basic Procedure

molecules composition acids support

In electrophoresis, an electric field is generated to separate charged molecules that are suspended in a matrix or gel support. Negatively charged molecules move toward the anode, on one side of the gel, and positively charged molecules move toward the cathode, on the other side. The gel itself is a porous matrix, or meshwork, often made of carbohydrate chains. Molecules are pulled through the open spaces in the gel, but they are slowed down by the meshwork based on their differing properties.

A solution containing DNA is added to the gel using a micropipette.

The parameters that determine the migration rate of these molecules through the meshwork are the strength of the electric field, the composition of the gel support or matrix, the composition of the liquid buffer solution the gel sits in, and the size, shape, charge, and chemical composition of the molecules being separated. Smaller molecules move faster than larger molecules, because they encounter less frictional drag in the gel. The size of the pores in the gel can be changed so this frictional drag is increased or decreased, allowing faster separation, or finer resolution.

The electrophoretic technique can analyze and purify a variety of bio-molecules, but is mainly used to separate nucleic acids and proteins. A basic consideration for choosing this technique is the composition of the sample to be separated—for example, does it contain nucleic acids (DNA or RNA), or is it composed of proteins, or carbohydrates? What are the sizes of the molecules to be separated? Another important consideration is the purpose of the separation—is it qualitative, where the technique is being used to evaluate the composition of the sample, or is it quantitative, in that the separated materials are to be collected for further analysis? Cellulose or starch is used as a support medium for low molecular-weight biomolecules such as amino acids and carbohydrates, whereas separation of proteins and nucleic acids are almost always done in gels made of a porous insoluble material such as agarose or acrylamide.

Gel Electrophoresis - Separation Of Proteins [next]

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