Electrophoresis
Electrophoresis
Electrophoresis is a technique used for the separation of biological molecules based on their movement due to the influence of a direct electric current. The technique was pioneered in 1937 by the Swedish chemist Arne Tiselius for the separation of proteins. It has now been extended to the separation of many other different classes of biomolecules including nucleic acids, carbohydrates and amino acids.
Electrophoresis has become increasingly important in the laboratory for basic research, biomedical research and in clinical settings for the diagnosis of disease. Electrophoresis is not commonly used to purify proteins in large quantities because other methods exist which are simpler, faster, and more efficient. However, it is valuable as an analytical technique for detecting and quantifying minute traces of many bio-molecules in a mixture. It is also useful for determining certain physical properties such as molecular weight, isoelectric point, and biological activity.
In paper electrophoresis, the charged molecules (i.e., proteins) to be separated are electrophoresed upward through a buffersolution toward electrodes immersed on either side of a U-shaped tube. The individual proteins are resolved because they have different mobilities as described above. This technique separates the biomolecules on the basis of their charges. Positively charged molecules migrate toward the negative electrode (cathode) and negatively charged particles move to the positive electrode (anode). The migration of the particles to the electrodes can be followed by instruments which measure the refractive index or absorption of light by the solution.
Another type of electrophoresis is gel electrophoresis. Here, the sample is loaded into a gel matrix support. This has many important advantages:
1. The density and size of the holes (pores) of gels can be easily controlled and adjusted for different biomolecules or different experimental conditions. Since the gel pore size is on the order of the dimensions of the macromolecules, the separations are based on molecular sieving as well as electrophoretic mobility of the molecules. Large molecules migrate more slowly relative to smaller molecules as they cannot pass as easily through the gel during electrophoresis.
In addition to proteins, DNA fragments can be separated from one another electrophoretically. Highly purified agarose, a major component of seaweed, is used as the solid gel matrix into which the DNA samples are loaded for electrophoresis. By varying the agarose concentration in the gel, DNA fragments in different size ranges can be separated.
The agarose is dissolved in water, heated and cast as a gel slab approximately 0.2 in (0.5 cm) in thickness. Wells are formed at one end of the gel for the loading of the DNA sample. The slab is then placed horizontally into the electrophoresis buffer chamber. The DNA migrates in bands toward the positive electrode. The smaller molecules pass through the matrix more rapidly than the larger ones, which are restricted. The DNA bands are then stained using a fluorescent dye such as ethidium bromide. The stained gel is then viewed directly under ultraviolet light and photographed.
Because proteins are typically much smaller than DNA, they are run in gels containing polymers of much smaller pore size. The most common technique for protein separation is known as SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). in this approach the proteins are treated with a detergent (sodium dodecyl sulfate) which unfolds them and gives them similar shape and ratio of charge to mass. Thus, proteins, treated with SDS separate on the basis of mass, with the smaller proteins migrating more rapidly through the gel. The gel matrix is polyacrylamide, a synthetic copolymer which has excellent molecular sieving properties. Polyacrylamide gels are cast between glass plates to form what is called a “sandwich.” Protein gels are
KEY TERMS
Biological activity— The specific biochemical function of a biomolecule such as enzyme affinity for a substrate.
Biomolecule— An organic compound present in living organisms.
Electrophoresis gel— A natural or synthetic polymer matrix used as a support for sample separation in electrophoresis.
Isoelectric point— The pH at which a molecule has no electric charge and does not move in an electric field.
Molecular sieving— The restriction of molecules migrating through a gel due to their size.
Sodium dodecyl sulfate (SDS)— A negatively charged detergent which strongly binds to amino acid residues of protein and denatures the protein.
generally run in a vertical fashion. The electrophoresis apparatus has an upper and lower buffer tank. The top and bottom of the sandwich is in contact with either buffer. Protein is loaded into the wells in the upper buffer tank and current is applied. The proteins thus migrate down through the gel in bands, according to their sizes. After electrophoresis, the polyacrylamide gel is removed from between the glass plates and chemically stained to show protein bands which can then be studied. The SDS-PAGE technique allows researchers to study parts of proteins and protein-protein interactions. If a protein has different subunits they will be separated by SDS treatment and will form separate bands.
Electrophoretic techniques have also been adapted to other applications such as the determination of protein isoelectric points. Affinity gels with biospecific properties are used to study binding sites and surface features of proteins. Continuous flow electrophoresis is applied to separations in free solution and has found very useful application in blood cell separation. Recently, High Performance Capillary Electrophoresis (HPCE) has been developed for the separation of many classes of biological molecules.
Resources
BOOKS
Petersen, John R. and Amin A. Mohammad. Clinical and Forensic Applications of Capillary Electrophoresis. Totowa, NJ: Humana Press, 2001.
Westermeier, Reiner. Electrophoresis in Practice: A Guide to methods and Applications of DNA and Protein Separations. New York: John Wiley & Sons, 2005.
Leonard D. Holmes