Gel electrophoresis 12582 222903571 2008-07-01T17:51:23Z 137.122.32.44 /* History */ {{redirect|PAGE||Page}} {{Infobox chemical analysis | name = Gel electrophoresis | image = Gel electrophoresis apparatus.JPG | caption ='''Gel electrophoresis apparatus''' - An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera. | acronym = | classification =[[Electrophoresis]] | analytes = | manufacturers = | related = [[Capillary electrophoresis]]<br>[[SDS-PAGE]]<br>[[Two-dimensional gel electrophoresis]]<br>[[Temperature gradient gel electrophoresis]] | hyphenated = }} '''Gel electrophoresis''' is a technique used for the separation of [[DNA|deoxyribonucleic acid]], [[RNA|ribonucleic acid]], or [[protein]] [[molecule]]s using an electric current applied to a gel matrix.<ref name=Stryer>{{cite book | author = Berg JM, Tymoczko JL Stryer L | title = Molecular Cell Biology | edition = 5th ed. | publisher = WH Freeman | year = 2002 | url = http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=stryer.section.438#455 | isbn = 0-7167-4955-6}}</ref> It is usually performed for analytical purposes, but may be used as a preparative technique prior to use of other methods such as [[mass spectrometry]], [[Restriction fragment length polymorphism|RFLP]], [[Polymerase chain reaction|PCR]], [[cloning]], [[DNA sequencing]], or [[Southern blot]]ting for further characterization. ==Separation== The term "[[gel]]" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases the gel is a [[crosslinked polymer]] whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating [[protein]]s or small [[nucleic acid]]s ([[DNA]], [[RNA]], or [[oligonucleotide]]s) the gel is usually composed of different concentrations of [[acrylamide]] and a [[cross-linker]], producing different sized mesh networks of [[polyacrylamide]]. When separating larger nucleic acids (greater than a few hundred [[base (chemistry)|bases]]), the preferred matrix is purified [[agarose]]. In both cases, the gel forms a solid, yet porous matrix. [[Acrylamide]], in contrast to [[polyacrylamide]], is a [[neurotoxin]] and must be handled using appropriate safety precautions to avoid poisoning. "[[Electrophoresis]]" refers to the [[electromotive force]] (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, usually determined by mass, toward the positive [[anode]] if negatively charged or toward the negative [[cathode]] if positively charged <ref>{{cite book | last = Robyt | first = John F. | last2 = White | first2 = Bernard J. | title = Biochemical Techniques Theory and Practice | place= Illinois | publisher = Waveland Press | year = 1990 | isbn = 0-88133-556-8}}</ref>. ==Visualization== [[Image:AgaroseGel.jpg|thumb|left|'''I Agarose gel prepared for DNA analysis''' - The first lane contains a ''DNA ladder'' for sizing, and the other four lanes show variously-sized DNA fragments that are present in some but not all of the samples.]] After the electrophoresis is complete, the molecules in the gel can be [[staining (biology)|stained]] to make them visible. [[Ethidium bromide]], silver, or [[coomassie]] blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules [[fluorescence|fluoresce]] under [[ultraviolet]] light, a [[photograph]] can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain [[radioactivity]] added for visibility, an [[isotopic tracer|autoradiogram]] can be recorded of the gel. If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components. Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are [[molecular weight size marker]]s available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule. ==Applications== Gel electrophoresis is used in [[Forensic chemistry|forensics]], [[molecular biology]], [[genetics]], [[microbiology]] and [[biochemistry]]. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software. Depending on the type of analysis being performed, other techniques are often implemented in conjunction with the results of gel electrophoresis, providing a wide range of field-specific applications. ===Nucleic acids=== In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the naturally-occurring negative charge carried by their [[sugar]]-[[phosphate]] backbone.<ref name=Lodish>{{cite book | author = Lodish H, Berk A, Matsudaira P, ''et al'' | title = Molecular Cell Biology | edition = 5th ed. | publisher = WH Freeman: New York, NY | year = 2004 | url = http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=mcb.section.1637#1648 | isbn = 978-0716743668}}</ref> Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their [[radius of gyration]], or, for non-cyclic fragments, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the [[hydrogen bond]]s, such as [[sodium hydroxide]] or [[formamide]], are used to denature the nucleic acids and cause them to behave as long rods again.<ref>Troubleshooting DNA agarose gel electrophoresis. Focus 19:3 p.66 (1997).</ref> Gel electrophoresis of large [[DNA]] or [[RNA]] is usually done by [[agarose gel electrophoresis]]. See the "[[Chain termination method]]" page for an example of a [[polyacrylamide]] DNA sequencing gel. ===Proteins=== [[Image:SDSPAGE.png|thumb|right|'''SDS-PAGE [[autoradiography]]''' - The indicated proteins are present in different concentrations in the two samples.]] Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually [[Denaturation (biochemistry)|denatured]] in the presence of a [[detergent]] such as [[sodium dodecyl sulfate]]/[[sodium dodecyl phosphate]] (SDS/SDP) that coats the proteins with a negative charge.<ref name=Stryer /> Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.<ref name=Stryer /> [[Protein]]s are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ([[SDS-PAGE]]), by [[Native Gel Electrophoresis|native gel electrophoresis]], by quantitative preparative native continuous polyacrylamide gel electrophoresis ([[QPNC-PAGE]]), or by [[2-D electrophoresis]]. ==History== *1930s - first reports of the use of [[sucrose]] for gel electrophoresis *1955 - introduction of [[starch]] gels, mediocre separation *1959 - introduction of [[acrylamide]] gels (Raymond and Weintraub); accurate control of parameters such as pore size and stability *1964 - disc gel electrophoresis (Ornstein and Davis) *1969 - introduction of [[Denaturation (biochemistry)|denaturing]] agents especially [[Sodium dodecyl sulfate|SDS]] separation of [[protein]] subunit (Beber and Osborn) *1970 - Laemmli separated 28 components of [[T4 phage]] using a stacking gel and SDS *1975 - 2-dimensional gels (O’Farrell); [[isoelectric focusing]] then SDS gel electrophoresis *1977 - [[DNA sequencing|sequencing]] gels *late 1970s - [[agarose]] gels *1983 - [[pulsed field gel electrophoresis]] enables separation of large DNA molecules *1983 - introduction of [[capillary electrophoresis]] A 1959 book on electrophoresis by Milan Bier cites references from the 1800s.<ref>{{cite book | author = Milan Bier (ed.) | title = Electrophoresis. Theory, Methods and Applications | publisher = Academic Press | pages = 225 | edition = 3rd printing | year = 1959 | oclc = 1175404 | id = LCC 59-7676}}</ref> However, [[Oliver Smithies]] made significant contributions. Bier states: "The method of Smithies ... is finding wide application because of its unique separatory power." Taken in context, Bier clearly implies that Smithies' method is an improvement. ==See also== * [[DNA electrophoresis]] * [[Electrofocusing]] * [[Gel isolation]] * [[Northern blot]]ting * [[Protein electrophoresis]] * [[Southern blot]]ting * [[Western blot]]ting * [[Zymography]] * [[Native Gel Electrophoresis]] ==References== <!-- --------------------------------------------------------------- See http://en.wikipedia.org/wiki/Wikipedia:Footnotes for a discussion of different citation methods and how to generate footnotes using the <ref> & </ref> tags and the {{Reflist}} template -------------------------------------------------------------------- --> {{Reflist}} ==External links== * [http://gslc.genetics.utah.edu/units/biotech/gel Biotechniques Laboratory electrophoresis demonstration], from the University of Utah's Genetic Science Learning Center * [http://www3.interscience.wiley.com/cgi-bin/abstract/113343976/ABSTRACT Discontinuous native protein gel electrophoresis] {{Protein methods}} [[Category:Protein methods]] [[Category:Molecular biology]] [[Category:Laboratory techniques]] [[Category:Electrophoresis]] [[Category:Polymerase chain reaction]] [[ar:فصل كهربائي للهلام]] [[ca:Gel d'electroforesi]] [[de:Gelelektrophorese]] [[es:Electroforesis en gel]] [[id:Elektroforesis gel]] [[nl:Gelelektroforese]] [[pt:Eletroforese em gel]] [[ur:ھلامہ برقی رحلان]] [[zh:凝膠電泳]]