- Electrophoresis is the term used to describe the motion of particles in a gel or fluid within a relatively uniform electric field.
- Electrophoresis may be used to separate molecules based on charge, size, and binding affinity.
- The technique is mainly applied to separate and analyze biomolecules, such as DNA, RNA, proteins, nucleic acids, plasmids, and fragments of these macromolecules.
- Electrophoresis is one of the techniques used to identify source DNA, as in paternity testing and forensic science.
Electrophoresis of anions or negatively charged particles is called anaphoresis. Electrophoresis of cations or positively charged particles is called cataphoresis.
Electrophoresis was first observed in 1807 by Ferdinand Frederic Reuss of Moscow State University, who noticed clay particles migrated in water subjected to a continuous electric field.
How Electrophoresis Works
In electrophoresis, there are two primary factors that control how quickly a particle can move and in what direction. First, the charge on the sample matters. Negatively charged species are attracted to the positive pole of an electric field, while positively charged species are attracted to the negative end. A neutral species may be ionized if the field is strong enough. Otherwise, it doesn’t tend to be affected.
The other factor is particle size. Small ions and molecules can move through a gel or liquid much more quickly than larger ones.
While a charged particle is attracted to an opposite charge in an electric field, there are other forces that affect how a molecule moves. Friction and the electrostatic retardation force slow the progress of particles through the fluid or gel. In the case of gel electrophoresis, the concentration of the gel can be controlled to determine the pore size of the gel matrix, which influences mobility. A liquid buffer is also present, which controls the pH of the environment.
As molecules are pulled through a liquid or gel, the medium heats up. This can denature the molecules as well as affect the rate of movement. The voltage is controlled to try to minimize the time required to separate molecules, while maintaining a good separation and keeping the chemical species intact. Sometimes electrophoresis is performed in a refrigerator to help compensate for the heat.
Types of Electrophoresis
Electrophoresis encompasses several related analytical techniques. Examples include:
- affinity electrophoresis – Affinity electrophoresis is a type of electrophoresis in which particles are separated based on complex formation or biospecific interaction
- capillary electrophoresis – Capillary electrophoresis is a type of electrophoresis used to separate ions depending mainly on the atomic radius, charge, and viscosity. As the name suggests, this technique is commonly performed in a glass tube. It yields quick results and a high resolution separation.
- gel electrophoresis – Gel electrophoresis is a widely used type of electrophoresis in which molecules are separated by movement through a porous gel under the influence of an electrical field. The two main gel materials are agarose and polyacrylamide. Gel electrophoresis is used to separate nucleic acids (DNA and RNA), nucleic acid fragments, and proteins.
- immunoelectrophoresis – Immunoelectrophoresis is the general name given to a variety of electrophoretic techniques used to characterize and separate proteins based on their reaction to antibodies.
- electroblotting – Electroblotting is a technique used to recover nucleic acids or proteins following electrophoresis by transferring them onto a membrane. The polymers polyvinylidene fluoride (PVDF) or nitrocellulose are commonly used. Once the specimen has been recovered, it can be further analyzed using stains or probes. A western blot is one form of electroblotting used to detect specific proteins using artificial antibodies.
- pulsed-field gel electrophoresis – Pulsed-field electrophoresis is used to separate macromolecules, such as DNA, by periodically changing the direction of the electric field applied to a gel matrix. The reason the electric field is changed is because traditional gel electrophoresis is unable to efficiently separate very large molecules that all tend to migrate together. Changing the direction of the electric field gives the molecules additional directions to travel, so they have a path through the gel. The voltage is generally switched between three directions: one running along the axis of the gel and two at 60 degrees to either side. Although the process takes longer than traditional gel electrophoresis, it’s better at separating large pieces of DNA.
- isoelectric focusing – Isoelectric focusing (IEF or electrofocusing) is a form of electrophoresis that separates molecules based on different isoelectric points. IEF is most often performed on proteins because their electrical charge depends on pH.