Electrophoresis

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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 (?), is defined as ? = 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:

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.
Gels are easy to chemically modify, for related techniques such as affinity electrophoresis which separates biomolecules on the basis of biomolecular affinity or recognition.
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.

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

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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.