Editing Gel electrophoresis (section)

==Physical basis==
{{See also|Electrophoresis}}
[[Image:SDS-PAGE_Electrophoresis.png|thumb|500px|Overview of gel electrophoresis.]]
Electrophoresis is a process that enables the sorting of molecules based on charge, size, or shape. Using an electric field, molecules such as DNA can be made to move through a gel made of [[agarose]] or [[polyacrylamide]]. The electric field consists of a negative charge at one end which pushes the molecules through the gel and a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric field is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel.<ref name="Wilson 2018">{{cite book | last=Wilson | first=Keith | title=Wilson and Walker's principles and techniques of biochemistry and molecular biology | publisher=Cambridge University Press | publication-place=Cambridge, United Kingdom New York, NY | year=2018 | isbn=978-1-316-61476-1 | oclc=998750377 | page=}}</ref>

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 are 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)|base]]s), 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. Agarose is composed of long unbranched chains of uncharged carbohydrates without cross-links, resulting in a gel with large pores allowing for the separation of macromolecules and [[affinity electrophoresis|macromolecular complexes]].<ref name="Boyer 2000">{{cite book | last=Boyer | first=Rodney | title=Modern experimental biochemistry | publisher=Benjamin Cummings | publication-place=San Francisco | year=2000 | isbn=978-0-8053-3111-0 | oclc=44493241 | language=et | page=}}</ref>

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 field, the molecules will move through the matrix at different rates, determined largely by their mass when the charge-to-mass ratio (Z) of all species is uniform. However, when charges are not uniform, the electrical field generated by the electrophoresis procedure will cause the molecules to migrate differentially according to charge. Species that are net positively charged will migrate towards the [[cathode]] (which is negatively charged because this is an [[Electrolytic cell|electrolytic]] rather than [[galvanic cell]]), whereas species that are net negatively charged will migrate towards the positively charged anode. Mass remains a factor in the speed with which these non-uniformly charged molecules migrate through the matrix toward their respective electrodes.<ref name="Robyt 1990">{{cite book | last=Robyt | first=John | title=Biochemical techniques : theory and practice | publisher=Waveland Press | publication-place=Prospect Heights, Ill | year=1990 | isbn=978-0-88133-556-9 | oclc=22549624 | page=}}</ref>

If several samples have been loaded into adjacent wells in the gel, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows the 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 indistinguishable smears representing multiple unresolved components. {{citation needed|date=January 2011}}  Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel at 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 to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule. (Equivalently, the distance traveled is inversely proportional to the log of the samples's molecular weight).<ref name="pmid22546956">{{cite journal| author1=Lee PY| author2=Costumbrado J| author3=Hsu CY| author4=Kim YH| title=Agarose gel electrophoresis for the separation of DNA fragments. | journal=J Vis Exp | year= 2012 | volume=  | issue= 62 | pages=  | pmid=22546956 | doi=10.3791/3923 | pmc=4846332 }}</ref>

There are limits to electrophoretic techniques. Since passing a current through a gel causes heating, gels may melt during electrophoresis. Electrophoresis is performed in buffer solutions to reduce pH changes due to the electric field, which is important because the charge of DNA and RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution. There are also limitations in determining the molecular weight by SDS-PAGE, especially when trying to find the MW of an unknown protein. Certain biological variables are difficult or impossible to minimize and can affect electrophoretic migration. Such factors include protein structure, post-translational modifications, and amino acid composition. For example, tropomyosin is an acidic protein that migrates abnormally on SDS-PAGE gels. This is because the acidic residues are repelled by the negatively charged SDS, leading to an inaccurate mass-to-charge ratio and migration.<ref>{{Cite web|url=https://www.bio-rad.com/sites/default/files/webroot/web/pdf/lsr/literature/Bulletin_3133.pdf |archive-url=https://web.archive.org/web/20211117045850/https://www.bio-rad.com/sites/default/files/webroot/web/pdf/lsr/literature/Bulletin_3133.pdf |archive-date=2021-11-17 |url-status=live |title=Molecular Weight Determination by SDS-PAGE, Rev B|website = www.bio-rad.com| accessdate=2022-03-23}}</ref> Further, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.