Electrophoresis is a standard technique used for various purposes worldwide for DNA, RNA, and protein analysis. It has been known for nearly a century and various techniques have been developed over decades. The basic principle is based on the separation of proteins through application of an electric field across the compartment filled with medium of different pH. Iscotachophoresis or ion migration method was the first fundamental electrophoresis principle for separation of acids and metals. With the introduction of this method, it was clear that when an electric field is applied, the charged components are separated based on differences in their electrophoretic mobilities.
Gel electrophoresis is a type of electrophoresis that is widely used and has several types. Hydrated gel networks have many desirable properties for electrophoresis as they allow a wide variety of mechanically stable experimental formats such as horizontal and vertical electrophoresis in slab gels or electrophoresis in tubes and capillaries. The mechanical stability facilitates post electrophoretic manipulation making further experimentation such as blotting, electroelution or MS identification / finger printing of intact proteins or also of proteins digested in gel slices. Because gels used in biochemistry are chemically unreactive, they minimally interact with biomolecules during electrophoresis, thereby allowing the separation based on physical rather than chemical differences between samples. One of the most important aspects of gel electrophoresis technique is staining. Once sample molecules have separated in the gel matrix, it is crucial to visualize their position which can be achieved by staining with an agent appropriate for the sample. The most commonly used gels for separation of nucleic acids and proteins are agarose gels and acrylamide gels. Sometimes, starch gels are also used.
Agarose: It is a polymer composed of a repeating disaccharide unit called agarobiose which consists of galactose and 3,6-anhydrogalactose. It gives a more uniform degree of porosity than starch and this may be varied by altering the initial concentration of the suspension, low concentration gives large pores whereas high concentration gives smaller pores. This gel has found widespread use especially in the separation of DNA molecules. It may also be used in electrophoretic procedures involving protein samples such as immuno-electrophoresis. The charge is evenly distributed in nucleic acids, which makes it possible to accurately determine DNA molecular masses based on their mobility in agarose gels. The mechanical stability of agarose is limited, while it is sufficient to form a stable horizontal gel, it compromises the possibilities for post-electrophoretic manipulation.
Acrylamide: It is much stronger and is suitable for electrophoretic separation of both proteins and nucleic acids. The inclusion of a small amount of acrylamide cross linked by a methylene bridge permits the formation of a cross linked gel with a highly controlled porosity which is also mechanically strong and chemically inert. Such gels are suitable for high resolution separation of DNA and proteins across a large mass range.