Nov 21, 2008
Protein electrophoresis is a sensitive analytical form of chromatography that allows the separation of charged molecules in a solution medium under the influence of an electric field. A wide range of molecules may be separated by electrophoresis, including, but not limited to DNA, RNA, and protein molecules.
The degree of separation and rate of molecular migration of mixtures of molecules depends upon the size and shape of the molecules, the respective molecular charges, the strength of the electric field, the type of medium used (e.g., cellulose acetate, starch gels, paper, agarose, polyacrylamide gel, etc.) and the conditions of the medium (e.g., electrolyte concentration, pH, ionic strength, viscosity, temperature, etc.).
Some mediums (also known as support matrices) are porous gels that can also act as a physical sieve for macromolecules.
In general, the medium is mixed with buffers needed to carry the electric charge applied to the system. The medium/buffer matrix is placed in a tray. Samples of molecules to be separated are loaded into wells at one end of the matrix. As electrical current is applied to the tray, the matrix takes on this charge and develops positively and negatively charged ends. As a result, molecules such as DNA and RNA that are negatively charged, are pulled toward the positive end of the gel.
Because molecules have differing shapes, sizes, and charges they are pulled through the matrix at different rates and this, in turn, causes a separation of the molecules. Generally, the smaller and more charged a molecule, the faster the molecule moves through the matrix.
When DNA is subjected to electrophoresis, the DNA is first broken by what are termed restriction enzymes that act to cut the DNA is selected places. After being subjected to restriction enzymes, DNA molecules appear as bands (composed of similar length DNA molecules) in the electrophoresis matrix. Because nucleic acids always carry a negative charge, separation of nucleic acids occurs strictly by molecular size.
Proteins have net charges determined by charged groups of amino acids from which they are constructed. Proteins can also be amphoteric compounds, meaning they can take on a negative or positive charge depending on the surrounding conditions. A protein in one solution might carry a positive charge in a particular medium and thus migrate toward the negative end of the matrix. In another solution, the same protein might carry a negative charge and migrate toward the positive end of the matrix. For each protein there is an isoelectric point related to a pH characteristic for that protein where the protein molecule has no net charge. Thus, by varying pH in the matrix, additional refinements in separation are possible.
The advent of electrophoresis revolutionized the methods of protein analysis. Swedish biochemist Arne Tiselius was awarded the 1948 Nobel Prize in chemistry for his pioneering research in electrophoretic analysis. Tiselius studied the separation of serum proteins in a tube (subsequently named a Tiselius tube) that contained a solution subjected to an electric field.
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis techniques pioneered in the 1960s provided a powerful means of protein fractionation (separation). Because the protein bands did not always clearly separate (i.e., there was often a great deal of overlap in the protein bands) only small numbers of molecules could be separated. The subsequent development in the 1970s of a two-dimensional electrophoresis technique allowed greater numbers of molecules to be separated.
Two-dimensional electrophoresis is actually the fusion of two separate separation procedures. The first separation (dimension) is achieved by isoelectric focusing (IEF) that separates protein polypeptide chains according to amino acid composition. IEF is based on the fact that proteins will, when subjected to a pH gradient, move to their isoelectric point. The second separation is achieved via SDS slab gel electrophoresis that separates the molecule by molecular size. Instead of broad, overlapping bands, the result of this two-step process is the formation of a two-dimensional pattern of spots, each comprised of a unique protein or protein fragment. These spots are subsequently subjected to staining and further analysis.
Some techniques involve the application of radioactive labels to the proteins. Protein fragments subsequently obtained from radioactively labels proteins may be studied my radiographic measures.
There are many variations on gel electrophoresis with wide-ranging applications. These specialized techniques include Southern, Northern, and Western blotting. Blots are named according to the molecule under study. In Southern blots, DNA is cut with restriction enzymes then probed with radioactive DNA. In Northern blotting, RNA is probed with radioactive DNA or RNA. Western blots target proteins with radioactive or enzymatically tagged antibodies.
Modern electrophoresis techniques now allow the identification of homologous DNA sequences and have become an integral part of research into gene structure, gene expression, and the diagnosis of heritable and autoimmune diseases. Electrophoretic analysis also allows the identification of bacterial and viral strains and is finding increasing acceptance as a powerful forensic tool.
©2000-2008
Enotes.com Inc.
All Rights Reserved