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 biomolecules in a mixture. It is also useful for determining certain physical properties such as molecular weight , isoelectric point, and biological activity.

Electrophoretic theory

Electrophoretic separations are based upon the fact that the electrical force (F) on a charged particle (ion) in an electrical field (E) is proportional to the charge of the particle (q), or F = qE (Eq 1).

The migration of the charged particle in the electric field, called the electrophoretic mobility (μ), is defined as μ = v/E = q/f (Eq 2), where v is the velocity of the charged particle and f is a complex term called the frictional coefficient . The frictional coefficient relates to the size and the shape of the particle. From equation (2) it can be seen that electrophoretic mobility decreases for larger particles and increases with higher charge.

Methodology and applications

The electrophoresis equipment can have several designs. The simplest approach is the moving boundary technique (Figure 3). The charged molecules (i.e., proteins) to be separated are electrophoresed upward through a buffer solution 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.

The most important class of electrophoresis is zone electrophoresis. In zone electrophoresis, the sample to be separated is applied to a solid matrix through which it migrates under the force of an applied electric potential. Two major classes of zone electrophoresis will be discussed below.

Gel electrophoresis

The sample is loaded into a gel matrix support. This has many important advantages:

1. The density and porosity 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 are retarded relative to smaller molecules as they cannot pass as easily through the gel during electrophoresis.
2. Gels are easy to chemically modify, for related techniques such as affinity electrophoresis which separates biomolecules on the basis of biomolecular affinity or recognition.
3. Excellent separation power.

Gel electrophoresis of DNA

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 (Figure 2).

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.

Gel electrophoresis of proteins

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) (Figure 1). 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 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.

Paper electrophoresis

This technique is useful for the separation of small charged molecules such as amino acids and small proteins. A strip of filter paper is moistened with buffer and the ends of the strip are immersed into buffer reservoirs containing the electrodes. The samples are spotted in the center of the paper, high voltage is applied, and the spots migrate according to their charges. After electrophoresis, the separated components can be detected by a variety of staining techniques, depending upon their chemical identity.

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.

See also Bioassay; Biochemistry; Biotechnology; Proteomics.

Resources

books

Lodish, H., et al. Molecular Cell Biology. 4th ed. New York: W. H. Freeman & Co., 2000.

Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. 3rd ed. Worth Publishing, 2000.

Westheimer, Reiner. Electrophoresis in Practice. 3rd ed. New York: Springer Verlag, 2001.

organizations

Human Proteome Organization. <http://www.hupo.org/.>

Leonard D. Holmes

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.

Electrophoresis

Diseases caused by microorganisms are a threat to national security. Even in countries with well-developed healthcare systems, a massive outbreak can strain healthcare infrastructure. In other countries that are less wealthy and more politically volatile, the ravages of disease can sow the seeds of resentment against the more wealthy countries of the West. Thus, it is in a country's best interests to combat infectious diseases. One strategy is to examine the relevant microorganisms, particularly to find out the component(s) that are responsible for the infection. For many microbes, proteins are an important factor in the development of a disease. Proteins can function as receptors, to allow the microorganism to adhere to the surface of a host cell. As well, the toxins produced by microbes such as Escherichia coli O157:H7 and Vibrio chlorerae are proteins. Methods that can "dissect" microorganisms into their components, and which can compare a non-disease causing strain of a microbe to a diseasecausing strain to see what they differences are, is a valuable approach to fighting infectious disease. Electrophoresis is especially well suited to this role. Furthermore, specialized types of electrophoresis (i.e., pulsed field electrophoresis) allow the genetic material of the microorganism to be examined. Thus, electrophoresis can reveal much detail at the molecular level.

Electrophoresis is a sensitive analytical form of chromatography. Under the influence of an electrical field charged molecules can be separated from one another as they pass through a gel. The degree of separation and rate of molecular migration of mixtures of molecules depends upon a variety of factors, which can be tailored depending upon the intent of the separation. For example, conditions can be established that allow molecules of very large mass, but which differ from each other by only a fraction, to be visually separated. The factors that influence molecular separation include the individual size and shape of the molecules, their molecular charge, strength of the electric field, the type of support medium used (e.g., gels made of cellulose acetate, starch, paper, agarose, polyacrylamide) and the conditions of the medium (e.g., ion strength and concentration, pH, viscosity, temperature).

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.

In electrophoresis, the electric charge often is passed through what is known as a support medium. As summarized above, various support media can be used. They all share the trait that they are a three-dimensional arrangement of intertwined strands, which produces holes (or pores) through the gel matrix. Such matrices act as a physical sieve for macromolecules.

In general, the medium is mixed with a chemical mixture called a buffer. The buffer carries the electric charge that is applied to the system. The medium/buffer matrix is placed in a tray. Samples of molecules to be separated are loaded into wells or slots that have been formed 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 that are negatively charged such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein 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.

Intact DNA is so large that it cannot move through the pores of a gel (although the technique of pulsed field electrophoresis does allow very large pieces of DNA to be examined). When DNA is subjected to electrophoresis, the DNA is first cut into smaller pieces by restriction enzymes. Restriction enzymes recognize specific sequences of the building blocks of the DNA and cut the DNA at the particular site. There are many types of restriction enzymes, and so DNA can be cut into many different patterns. After electrophoresis, the pieces of DNA appear as bands (composed of similar length DNA molecules) in the electrophoresis matrix.

Proteins have net charges determined by charged groups of the amino acids from which they are constructed. Proteins can also be amphoteric compounds (a compound that 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 a pH in which the protein molecule has no net charge (the isoelectric point). By varying the pH in the matrix, additional refinements in separation are possible.

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis techniques pioneered in the 1960s provided a powerful means of protein separation. Still, because proteins of similar mass did not always clearly separate into discrete bands in the gel only small numbers of molecules could be separated.

The 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 the arrangement of amino acids that comprise a chain. 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, which 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.

Electrophoresis can be combined with the prior addition of a radioactive food source to the culture of bacteria. The bacteria will use the food to make new proteins, which will be radioactive. Following electrophoresis, the gel can be placed in contact with x-ray film. The radioactive bands or spots will register on the film, and so will determine what proteins were being made at the time of the experiment.

There are many other 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 DNA sequences that are the same, and have become an integral part of research into gene structure, gene expression, and the diagnosis of heritable diseases. Electrophoretic analysis also allows the identification of bacterial and viral strains and is finding increasing acceptance as a powerful forensic tool.

█ FURTHER READING:

BOOKS:

Birren, Bruce W., and Eric Hon Cheong Lai. Pulsed Field Electrophoresis: A Practical Guide. San Diego: Academic Press, 1997.

Rabilloud, Thierry. Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods (Principles and Practice). Berlin: Springer Verlag, 2000.

Westermeier, Reiner. Electrophoresis in Practice. Weinheim: Vch Verlagsgesellschaft 2001.

ELECTRONIC:

Colorado State University. "Gel Electrophoresis of DNA and RNA." Biomedical Hypertextbooks. January 15,2000. <http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/>(5 January 2003).

SEE ALSO

Chemical and Biological Detection Technologies
DNA Recognition Instruments
Microbiology: Applications to Espionage, Intelligence and Security
Thin Layer Chromatography

Electrophoresis

Resources

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

Electrophoresis

Electrophoresis is one of the most important techniques used by molecular biologists. To name only a few applications, deoxyribonucleic acid (DNA) electrophoresis is used to map the order of restriction fragments within chromosomes , to analyze DNA variation within a population by restriction fragment length polymorphisms (RFLPs), and to determine the nucleotide sequence of a piece of DNA.

Electrophoresis refers to the migration of a charged molecule through a restrictive matrix , or gel, drawn by an electrical force. As the force drags the molecule through the gel, it encounters resistance from the strands of the gel, retarding its rate of migration. In gel electrophoresis, larger molecules migrate more slowly than smaller ones, and so the distance of migration within a gel can be used to determine a molecule's size.

The rate of migration is inversely proportional to the logarithm of a molecule's size.

Although it is possible to separate whole chromosomes using specialized electrophoresis techniques, DNA that is to be analyzed by electrophoresis is usually cut into smaller pieces using restriction enzymes . Fragments of DNA prepared by treatment with restriction enzymes are commonly separated from one another, and their sizes determined, using a gel of agarose electrophoresis, a complex carbohydrate . DNA is negatively charged due to the phosphodiester bonds that join the individual nucleotide building blocks. DNA will therefore electrophorese toward the positive electrode when placed in an electrical field. To visualize the results after electrophoresis, the gel is soaked in a solution that causes DNA to fluoresce when exposed to ultraviolet light.

Treatment of the DNA sample with multiple restriction enzymes in various combinations enables the researcher to generate a restriction map of the original DNA fragment, which identifies the sites at the DNA where the restriction enzymes are.

Many research questions require a detailed analysis of one specific DNA fragment in a complex mixture. In such cases, a radioactive DNA probe can be used to identify the fragment based on its nucleotide sequence. The method, known as hybridization, is based on the rules of complementary base pairing (A bonds to T, G bonds to C). A probe is designed whose sequence is complementary to the piece of DNA to be detected. The gel-separated DNA is first transferred to a nylon membrane using a technique called a Southern blot.

Agarose, which is used to make electrophoresis gel, is derived from the seaweed agar.

During the blotting procedure, the strands within the DNA double helix are separated from each other, or denatured, by treatment with a base. Because double-stranded DNA is more stable than single-stranded, during the hybridization the single-stranded probe will locate and bind to the single-stranded gel-separated fragment with complementary sequence. Being fluorescent or radioactive, the position of the probe can be determined using photographic methods. The target sequence can then be removed by cutting at the piece of the gel that contains it.

The most common technique for determining DNA sequence is the Sanger method, which generates fragments that differ in length by a single nucleotide. High-resolution polyacrylamide gel electrophoresis is then used to separate the fragments and to allow the sequence to be determined.

SANGER, FREDERICK (1918–)

English biochemist who received two Nobel Prizes in chemistry. The first came in 1958, for finding the amino sequence of insulin, the protein that helps regulate blood sugar levels, and the second, in 1980, for inventing a technique to sequence the nucleotides in a strand of deoxyribonucleic acid (DNA).

Electrophoresis of ribonucleic acid (RNA) is an integral procedure in many studies of gene expression . RNA is isolated, separated by electrophoresis, and then the gel-separated RNA fragments are transferred to a nylon membrane using a technique called a Northern blot. Hybridization with a single-stranded DNA probe is then used to determine the position of a specific RNA fragment.

DNA and RNA are relatively simple in terms of structure and composition. Proteins , however, are composed of twenty different amino acids in various combinations, and proteins vary significantly in their three-dimensional structure. The composition of amino acids will affect the charge on the protein, which ultimately will affect its electrophoretic behavior. The shape of a protein similarly will affect its rate of migration. As a result, a specialized technique, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), is usually used to analyze proteins. In this method, protein samples are heated and then treated with the detergent sodium dodecyl sulfate (SDS). Proteins treated in this way are unfolded, linear, and uniformly coated by negatively charged detergent molecules. The rate of migration of treated proteins is inversely proportional to the logarithm of molecular weight. Following electrophoresis, the protein in the gel can be stained to visualize all the proteins in a sample, or the proteins in the gel can be transferred to a nylon membrane (Western blot) and specific ones detected with the use of enzyme -linked antibodies.

Regardless of the macromolecule being studied, gel electrophoresis is a crucial technique to the molecular biologist. Many scientific questions can be answered using electrophoresis, and as a result an active molecular biology research lab will have several benches that are devoted to the required specialized reagents and equipment.

see also DNA Sequencing

James E. Blankenship

Bibliography

Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York: Garland Publishing, 2000.

Stryer, Lubert. Biochemistry, 4th ed. New York: W. H. Freeman and Company, 1995.

Electrophoresis

Electrophoresis is a valuable approach to fighting infectious disease. Electrophoretic analysis allows the identification of bacterial and viral strains and is finding increasing acceptance as a powerful forensic tool.

Diseases caused by microorganisms are a threat to national security. One strategy is to examine the relevant microorganisms, particularly to find out the component(s) that are responsible for the infection. For many microbes, proteins are an important factor in the development of a disease. Proteins can function as receptors, to allow the microorganism to adhere to the surface of a host cell. As well, the toxins produced by microbes such as Escherichia coli O157:H7 and Vibrio chlorerae are proteins. Methods that can dissect microorganisms into their components, and that can compare a non-diseasecausing strain of a microbe to a disease-causing strain to see where they differ, are a valuable approach to fighting infectious disease. Electrophoresis is especially well suited to this role. Furthermore, specialized types of electrophoresis (i.e., pulsed field electrophoresis) allow the genetic material of the microorganism to be examined. Thus, electrophoresis can reveal much detail at the molecular level.

Electrophoresis is a sensitive analytical form of chromatography . Under the influence of an electrical field, charged molecules can be separated from one another as they pass through a gel. Gel electrophoresis is a method that separates macromolecules—either nucleic acids or proteins—on the basis of size, electric charge, and other physical properties. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means "to carry across." Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule's properties determine how rapidly an electric field can move the molecule through a gelatinous medium. Gel electrophoresis makes it possible to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid.

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.

In electrophoresis, the electric charge often is passed through one of various support mediums. In general, a medium is mixed with a chemical mixture called a buffer. The buffer carries the electric charge that is applied to the system. The medium/buffer matrix is placed in a tray with molecule samples to be separated. 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 that are negatively charged, such as deoxyribonucleic acid (DNA ), ribonucleic acid (RNA), and protein, are pulled toward the positive end of the gel.

Intact DNA is so large that it cannot move through the pores of a gel (although the technique of pulsed field electrophoresis does allow very large pieces of DNA to be examined). When DNA is subjected to electrophoresis, the DNA is first cut into smaller pieces by restriction enzymes. Restriction enzymes recognize specific sequences of the building blocks of the DNA and cut the DNA at the particular site. There are many types of restriction enzymes, and so DNA can be cut into many different patterns. After electrophoresis, the pieces of DNA appear as bands (composed of similar length DNA molecules) in the electrophoresis matrix.

Electrophoresis can be combined with the prior addition of a radioactive food source to the culture of bacteria. The bacteria will use the food to make new proteins, which will be radioactive. Following electrophoresis, the gel can be placed in contact with x-ray film. The radioactive bands or spots will register on the film, and so will determine what proteins were being made at the time of the experiment.

There are many other 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 DNA sequences that are the same. They have become an integral part of research into gene structure, gene expression, and the diagnosis of heritable diseases. Electrophoretic analysis also allows the identification of bacterial and viral strains and is finding increasing acceptance as a powerful forensic tool.

see also Chemical and biological detection technologies; Chromatography; DNA; DNA recognition instruments; DNA sequences, unique; DNA typing systems; Thin layer chromatography; Toxins.