Editing Gel electrophoresis

{{short description|Method for separation and analysis of biomolecules}}
{{Use dmy dates|date=October 2020}}

{{Infobox chemical analysis
| name = Gel electrophoresis
| image = Gel electrophoresis apparatus.JPG
| caption =Gel electrophoresis apparatus – an [[Agarose gel electrophoresis|agarose gel]] is placed in this buffer-filled box and an electric 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 positively charged anode (red wire). This occurs because phosphate groups found in the DNA fragments possess a negative charge which is repelled by the negatively charged cathode and are attracted to the positively charged anode.
| acronym = 
| classification =[[Electrophoresis]]
| analytes = 
| manufacturers = 
| related = [[Capillary electrophoresis]]<br>[[Polyacrylamide gel electrophoresis|SDS-PAGE]] <br>[[Two-dimensional gel electrophoresis]]<br>[[Temperature gradient gel electrophoresis]]
| hyphenated = 
}}

[[File: Gel Electrophoresis.svg|right|thumb|200px|The image above shows how small DNA fragments will migrate through agarose quickly but large size DNA fragments move more slowly during electrophoresis. The graph to the right shows the nonlinear relationship between the size of the DNA fragment and the distance migrated.]]

[[File:Gel Electrophoresis in DNA Fingerprinting.svg|right|thumb|200px|Gel electrophoresis is a process where an electric current is applied to DNA samples creating fragments that can be used for comparison between DNA samples.
<ol>
<li> DNA is extracted. </li>
<li> Isolation and amplification of DNA.  </li>
<li> DNA added to the gel wells.  </li>
<li> Electric current applied to the gel. </li>
<li> DNA bands are separated by size. </li>
<li> DNA bands are stained.  </li>
</ol>
]]

'''Gel electrophoresis''' is an [[electrophoresis]] method for separation and analysis of [[biomolecule|biomacromolecule]]s ([[DNA]], [[RNA]], [[protein]]s, etc.) and their fragments, based on their size and charge through a [[gel]]. It is used in [[clinical chemistry]] to separate proteins by charge or size (IEF agarose, essentially size independent) and in [[biochemistry]] and [[molecular biology]] to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments, or to separate proteins by charge.<ref name="pmid14507919">{{cite journal| author1=Kryndushkin DS| author2=Alexandrov IM| author3=Ter-Avanesyan MD| author4=Kushnirov VV| title=Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104. | journal=J Biol Chem | year= 2003 | volume= 278 | issue= 49 | pages= 49636–43 | pmid=14507919 | doi=10.1074/jbc.M307996200 | pmc= | doi-access=free }}</ref>

Nucleic acid molecules are separated by applying an [[electric field]] to move the negatively charged molecules through a gel matrix of [[agarose]], [[polyacrylamide]], or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.<ref name="Sambrook 2001">{{cite book | last=Sambrook | first=Joseph | title=Molecular cloning : a laboratory manual | publisher=Cold Spring Harbor Laboratory Press | publication-place=Cold Spring Harbor, N.Y | year=2001 | isbn=978-0-87969-576-7 | oclc=45015638 | language=es | page=}}</ref> Proteins are separated by the charge in agarose because the pores of the gel are too large to sieve proteins.  Gel electrophoresis can also be used for the separation of [[nanoparticle]]s.

Gel electrophoresis uses a gel as an anticonvective medium or sieving medium during electrophoresis. Gels suppress the thermal convection caused by the application of the electric field and can also serve to maintain the finished separation so that a post-electrophoresis stain can be applied.<ref name="Stryer">{{cite book | last=Berg | first=Jeremy | title=Biochemistry | publisher=W.H. Freeman | publication-place=New York | year=2002 | isbn=978-0-7167-4955-4 | oclc=48055706 | language=et | page=}}</ref> DNA gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via [[polymerase chain reaction]] (PCR), but may be used as a preparative technique for other methods such as [[mass spectrometry]], [[Restriction fragment length polymorphism|RFLP]], PCR, [[cloning]], [[DNA sequencing]], or [[southern blot]]ting for further characterization.

==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.

==Types of gel==

The types of gel most typically used are agarose and polyacrylamide gels. Each type of gel is well-suited to different types and sizes of the analyte.  Polyacrylamide gels are usually used for proteins and have very high resolving power for small fragments of DNA (5-500 bp). Agarose gels, on the other hand, have lower resolving power for DNA but a greater range of separation, and are therefore usually used for DNA fragments of 50–20,000 bp in size. (The resolution of over 6 Mb is possible with [[pulsed field gel electrophoresis]] (PFGE).)<ref>{{cite book |title=Molecular Cloning - A Laboratory Manual |author1=Tom Maniatis |author2=E. F. Fritsch |author3=Joseph Sambrook |edition=3rd |volume=1 |chapter=Chapter 5, protocol 1 |page=5.2–5.3  |isbn=978-0879691363 |year=1982 |publisher=Cold Spring Harbor Laboratory }}</ref> Polyacrylamide gels are run in a vertical configuration while agarose gels are typically run horizontally in a submarine mode. They also differ in their casting methodology, as agarose sets thermally, while polyacrylamide forms in a chemical polymerization reaction.

===Agarose===
[[File:Gel_electrophoresis_insert_comb.jpg|thumb|Inserting the gel comb in an agarose gel electrophoresis chamber]]

{{main|Agarose gel electrophoresis}}
Agarose gels are made from the natural [[polysaccharide]] [[polymer]]s extracted from [[seaweed]].
Agarose gels are easily cast and handled compared to other matrices because the gel setting is a physical rather than chemical change. Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.

Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kDa.<ref name="Smisek1989">{{cite journal | last1=Smisek | first1=David L. | last2=Hoagland | first2=David A. | title=Agarose gel electrophoresis of high molecular weight, synthetic polyelectrolytes | journal=Macromolecules | publisher=American Chemical Society (ACS) | volume=22 | issue=5 | year=1989 | issn=0024-9297 | doi=10.1021/ma00195a048 | pages=2270–2277| bibcode=1989MaMol..22.2270S }}</ref> Agarose gel electrophoresis can also be used for the separation of DNA fragments ranging from 50 [[base pair]] to several megabases (millions of bases),<ref>{{Cite journal |last=Voytas |first=Daniel |date=May 2001 |title=Agarose gel electrophoresis |url=https://pubmed.ncbi.nlm.nih.gov/18432695/ |journal=Current Protocols in Immunology |volume=Chapter 10 |pages=10.4.1–10.4.8 |doi=10.1002/0471142735.im1004s02 |issn=1934-368X |pmid=18432695 |s2cid=39623776 |access-date=1 March 2023 |archive-date=2 February 2022 |archive-url=https://web.archive.org/web/20220202131116/https://pubmed.ncbi.nlm.nih.gov/18432695/ |url-status=live }}</ref> the largest of which require specialized apparatus. The distance between DNA bands of different lengths is influenced by the percent agarose in the gel, with higher percentages requiring longer run times, sometimes days.  Instead high percentage agarose gels should be run with a [[Pulsed field gel electrophoresis|pulsed field electrophoresis]] (PFE), or [[field inversion electrophoresis]].

"Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications."<ref>{{cite web|title=Agarose gel electrophoresis (basic method)|url=http://www.methodbook.net/dna/agarogel.html|work=Biological Protocols|access-date=2022-03-23|archive-date=11 October 2018|archive-url=https://web.archive.org/web/20181011023503/http://www.methodbook.net/dna/agarogel.html|url-status=live}}</ref>

===Polyacrylamide===
{{main|Polyacrylamide gel electrophoresis}}

Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Pore size is controlled by modulating the concentrations of acrylamide and bis-acrylamide powder and by the [[QPNC-PAGE#Gel properties and polymerization time|polymerization time]] used in creating a gel. Care must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid and powdered forms.

Traditional [[DNA sequencing]] techniques such as [[Maxam–Gilbert sequencing|Maxam-Gilbert]] or [[Dideoxy termination#Chain termination method|Sanger]] methods used polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so the sequence could be read. Most modern DNA separation methods now use agarose gels, except for particularly small DNA fragments. It is currently most often used in the field of [[immunology]] and protein analysis, often used to separate different proteins or [[isoforms]] of the same protein into separate bands. These can be transferred onto a [[nitrocellulose]] or [[PVDF]] membrane to be probed with antibodies and corresponding markers, such as in a [[western blot]].

Typically [[resolving gels]] are made in 6%, 8%, 10%, 12% or 15%.  Stacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins, sample buffer, and ladders will be placed) is inserted.  The percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample. The smaller the known weight, the higher the percentage that should be used. Changes in the buffer system of the gel can help to further resolve proteins of very small sizes.<ref name="pmid17406207">{{cite journal | author=Schägger H | title=Tricine-SDS-PAGE. | journal=Nat Protoc | year=2006 | volume=1 | issue=1 | pages=16–22 | pmid=17406207 | doi=10.1038/nprot.2006.4 | pmc= | s2cid=209529082 | url=https://pubmed.ncbi.nlm.nih.gov/17406207 | access-date=23 March 2022 | archive-date=11 June 2022 | archive-url=https://web.archive.org/web/20220611043435/https://www.nature.com/articles/nprot.2006.4 | url-status=live }}</ref>

===Starch===

Partially [[hydrolysis|hydrolysed]] potato starch makes for another non-toxic medium for protein electrophoresis. The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be separated according to charge and size. They are visualised using Napthal Black or Amido Black staining. Typical starch gel concentrations are 5% to 10%.<ref name="Gordon1969">{{cite book | last=Gordon | first=A.H. | title=Electrophoresis of Proteins in Polyacrylamide and Starch Gels: Laboratory Techniques in Biochemistry and Molecular Biology | publisher=North-Holland Pub. Co | publication-place=Amsterdam | year=1969 | isbn=978-0-7204-4202-1 | oclc=21766 | page=}}</ref><ref name="pmid13276348">{{cite journal| author=Smithies O| title=Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. | journal=Biochem J | year= 1955 | volume= 61 | issue= 4 | pages= 629–41 | pmid=13276348 | doi=10.1042/bj0610629 | pmc=1215845 }}</ref><ref name="pmid5738223">{{cite journal | author1=Wraxall BG | author2=Culliford BJ | title=A thin-layer starch gel method for enzyme typing of bloodstains. | journal=J Forensic Sci Soc | year=1968 | volume=8 | issue=2 | pages=81–2 | pmid=5738223 | doi=10.1016/s0015-7368(68)70449-7 | pmc= | url=https://pubmed.ncbi.nlm.nih.gov/5738223 | access-date=23 March 2022 | archive-date=11 June 2022 | archive-url=https://web.archive.org/web/20220611043434/https://pubmed.ncbi.nlm.nih.gov/?Db=pubmed&Cmd=Retrieve&dopt=abstractplus&list_uids=5738223 | url-status=live }}</ref>

==Gel conditions==

===Denaturing===
[[File: TTGE profiles representing the bifidobacterial diversity of fecal samples journal pone 0050257 g004.png|thumb|400px|TTGE profiles representing the bifidobacterial diversity of fecal samples from two healthy volunteers (A and B) before and after AMC (Oral Amoxicillin-Clavulanic Acid) treatment]]

[[Denaturation (biochemistry)|Denaturing]] gels are run under conditions that disrupt the natural structure of the analyte, causing it to unfold into a linear chain.  Thus, the mobility of each [[macromolecule]] depends only on its linear length and its mass-to-charge ratio.  Thus, the secondary, tertiary, and quaternary levels of [[biomolecular structure]] are disrupted, leaving only the primary structure to be analyzed.

Nucleic acids are often denatured by including [[urea]] in the buffer, while proteins are denatured using [[sodium dodecyl sulfate]], usually as part of the [[Polyacrylamide gel electrophoresis|SDS-PAGE]] process.  For full denaturation of proteins, it is also necessary to reduce the covalent [[disulfide bond]]s that stabilize their [[tertiary structure|tertiary]] and [[quaternary structure]], a method called reducing PAGE. Reducing conditions are usually maintained by the addition of [[beta-mercaptoethanol]] or [[dithiothreitol]]. For a general analysis of protein samples, reducing PAGE is the most common form of [[protein electrophoresis]].

Denaturing conditions are necessary for proper estimation of molecular weight of RNA. RNA is able to form more intramolecular interactions than DNA which may result in change of its [[electrophoretic mobility]]. [[Urea]], [[Dimethyl sulfoxide|DMSO]] and [[glyoxal]] are the most often used denaturing agents to disrupt RNA structure. Originally, highly toxic [[methylmercury]] hydroxide was often used in denaturing RNA electrophoresis,<ref name="pmid632280">{{cite journal| author1=Buell GN| author2=Wickens MP| author3=Payvar F| author4=Schimke RT| title=Synthesis of full length cDNAs from four partially purified oviduct mRNAs. | journal=J Biol Chem | year= 1978 | volume= 253 | issue= 7 | pages= 2471–82 | pmid=632280 | doi= 10.1016/S0021-9258(17)38097-3| pmc= | doi-access=free }}</ref> but it may be method of choice for some samples.<ref name="pmid2570517">{{cite journal | author= Schelp C, Kaaden OR | title= Enhanced full-length transcription of Sindbis virus RNA by effective denaturation with methylmercury hydroxide. | journal= Acta Virol | year= 1989 | volume= 33 | issue= 3 | pages= 297–302 | pmid= 2570517 | doi=  | pmc=  | url= https://pubmed.ncbi.nlm.nih.gov/2570517 | access-date= 23 March 2022 | archive-date= 11 June 2022 | archive-url= https://web.archive.org/web/20220611043435/https://pubmed.ncbi.nlm.nih.gov/?Db=pubmed&Cmd=Retrieve&dopt=abstractplus&list_uids=2570517 | url-status= live }}</ref>

Denaturing gel electrophoresis is used in the DNA and RNA banding pattern-based methods [[temperature gradient gel electrophoresis]] (TGGE)<ref name="pmid12460271">{{cite journal| author1=Fromin N| author2=Hamelin J| author3=Tarnawski S| author4=Roesti D| author5=Jourdain-Miserez K| author6=Forestier N| display-authors=etal| title=Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns.| journal=Environ Microbiol| year=2002| volume=4| issue=11| pages=634–43| pmid=12460271| doi=10.1046/j.1462-2920.2002.00358.x| pmc=| bibcode=2002EnvMi...4..634F| url=https://pubmed.ncbi.nlm.nih.gov/12460271| access-date=23 March 2022| archive-date=11 June 2022| archive-url=https://web.archive.org/web/20220611043437/https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1046/j.1462-2920.2002.00358.x?sid=nlm%3Apubmed| url-status=live}}</ref> and denaturing gradient gel electrophoresis (DGGE).<ref name="pmid369706">{{cite journal | author1=Fischer SG | author2=Lerman LS | title=Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. | journal=Cell | year=1979 | volume=16 | issue=1 | pages=191–200 | pmid=369706 | doi=10.1016/0092-8674(79)90200-9 | pmc= | s2cid=9369012 | url=https://pubmed.ncbi.nlm.nih.gov/369706 | access-date=23 March 2022 | archive-date=11 June 2022 | archive-url=https://web.archive.org/web/20220611043438/https://www.cell.com/cell/pdf/0092-8674(79)90200-9.pdf?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2F0092867479902009%3Fshowall%3Dtrue | url-status=live }}</ref>

===Native===

[[File:Glucose-6-Phosphate Dehydrogenase activity stain.jpg|thumb|Specific enzyme-linked staining: [[Glucose-6-phosphate dehydrogenase deficiency|Glucose-6-Phosphate Dehydrogenase]] [[Isozyme|isoenzymes]] in ''[[Plasmodium falciparum]]'' infected [[Red blood cell]]s<ref name="pmid7012616">{{cite journal | author1=Hempelmann E | author2=Wilson RJ | title=Detection of glucose-6-phosphate dehydrogenase in malarial parasites. | journal=Mol Biochem Parasitol | year=1981 | volume=2 | issue=3–4 | pages=197–204 | pmid=7012616 | doi=10.1016/0166-6851(81)90100-6 | pmc= | url=https://pubmed.ncbi.nlm.nih.gov/7012616 | access-date=23 March 2022 | archive-date=6 July 2023 | archive-url=https://web.archive.org/web/20230706155553/https://www.sciencedirect.com/science/article/abs/pii/0166685181901006?via%3Dihub | url-status=live }}</ref>]]

Native gels are run in non-denaturing conditions so that the analyte's natural structure is maintained.  This allows the physical size of the folded or assembled complex to affect the mobility, allowing for analysis of all four levels of the biomolecular structure.  For biological samples, detergents are used only to the extent that they are necessary to [[lysis|lyse]] [[lipid bilayer|lipid membranes]] in the [[cell (biology)|cell]]. Complexes remain—for the most part—associated and folded as they would be in the cell. One downside, however, is that complexes may not separate cleanly or predictably, as it is difficult to predict how the molecule's shape and size will affect its mobility. These effects have been addressed by [[QPNC-PAGE|preparative native PAGE]].

Unlike denaturing methods, native gel electrophoresis does not use a charged [[Denaturation (biochemistry)|denaturing]] agent. The molecules being separated (usually [[proteins]] or [[nucleic acids]]) therefore differ not only in [[molecular mass]] and intrinsic charge, but also the cross-sectional area, and thus experience different electrophoretic forces dependent on the shape of the overall structure. For proteins, since they remain in the native state they may be visualized not only by general protein staining reagents but also by specific enzyme-linked staining.

A specific experiment example of an application of native gel electrophoresis is to check for enzymatic activity to verify the presence of the enzyme in the sample during protein purification. For example, for the protein alkaline phosphatase, the staining solution is a mixture of 4-chloro-2-2methylbenzenediazonium salt with 3-phospho-2-naphthoic acid-2'-4'-dimethyl aniline in Tris buffer. This stain is commercially sold as a kit for staining gels. If the protein is present, the mechanism of the reaction takes place in the following order: it starts with the de-phosphorylation of 3-phospho-2-naphthoic acid-2'-4'-dimethyl aniline by alkaline phosphatase (water is needed for the reaction). The phosphate group is released and replaced by an alcohol group from water. The electrophile 4- chloro-2-2 methylbenzenediazonium (Fast Red TR Diazonium salt) displaces the alcohol group forming the final product Red Azo dye. As its name implies, this is the final visible-red product of the reaction. In undergraduate academic experimentation of protein purification, the gel is usually run next to commercial purified samples to visualize the results and conclude whether or not purification was successful.<ref>{{Cite book|title = Fundamental Approaches to Biochemistry and Biotechnology|vauthors = Ninfa AJ, Ballou DP |publisher=Bethesda, Md: Fitzgerald Science Press|year=1998|isbn= 9781891786006}}</ref>

[[Native state|Native]] gel electrophoresis is typically used in [[proteomics]] and [[metallome|metallomics]]. However, native PAGE is also used to scan genes (DNA) for unknown mutations as in [[single-strand conformation polymorphism]].

==Buffers==
Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value.
These buffers have plenty of ions in them, which is necessary for the passage of electricity through them. Something like distilled water or benzene contains few ions, which is not ideal for the use in electrophoresis.<ref>{{Cite book|title=fundamental laboratory approaches for biochemistry and biotechnology|last1=Ninfa|first1=Alexander J.|last2=Ballou|first2=David P.|last3=Benore|first3=Marilee|publisher=Wiley|year=2009|isbn=978-0470087664|location=Hoboken, NJ|pages=161}}</ref> There are a number of buffers used for electrophoresis. The most common being, for nucleic acids [[TAE buffer|Tris/Acetate/EDTA]] (TAE), [[TBE buffer|Tris/Borate/EDTA]] (TBE). Many other buffers have been proposed, e.g. [[LB buffer|lithium borate (LB)]], (which is rarely used based on Pubmed citations), isoelectric histidine, pK matched Good's buffers, etc.; in most cases the purported rationale is lower current (less heat) matched ion mobilities, which leads to longer buffer life. Borate is problematic as borate can polymerize or interact with cis diols such as those found in RNA. TAE has the lowest buffering capacity, but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. LB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium borate).<ref name="pmid15351274">{{cite journal | author1=Brody JR | author2=Kern SE | title=History and principles of conductive media for standard DNA electrophoresis. | journal=Anal Biochem | year=2004 | volume=333 | issue=1 | pages=1–13 | pmid=15351274 | doi=10.1016/j.ab.2004.05.054 | pmc= | url=https://pubmed.ncbi.nlm.nih.gov/15351274 | access-date=23 March 2022 | archive-date=11 June 2022 | archive-url=https://web.archive.org/web/20220611043438/https://www.sciencedirect.com/science/article/abs/pii/S0003269704004932?via%3Dihub | url-status=live }}</ref>

Most SDS-PAGE protein separations are performed using a [[Gel electrophoresis of proteins#Buffer systems|"discontinuous" (or DISC) buffer system]] that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus on a single sharp band in a process called [[isotachophoresis]]. Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins.

==Visualization==
{{Further|Gel electrophoresis of nucleic acids#Visualization|Gel electrophoresis of proteins#Visualization}}

After the electrophoresis is complete, the molecules in the gel can be [[staining (biology)|stained]] to make them visible. DNA may be visualized using [[ethidium bromide]] which, when intercalated into DNA, [[fluorescence|fluoresce]] under [[ultraviolet]] light, while protein may be visualised using [[silver stain]] or [[Coomassie brilliant blue]] dye. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the molecules to be separated contain [[radioactivity]], for example in a [[DNA sequencing]] gel, an [[isotopic tracer|autoradiogram]] can be recorded of the gel.  [[Photograph]]s can be taken of gels, often using a [[gel doc]] system. Gels are then commonly labelled for presentation and scientific records on the popular figure-creation website, [https://sciugo.com SciUGo].

==Downstream processing==

After separation, an additional separation method may then be used, such as [[isoelectric focusing]] or [[SDS-PAGE]]. The gel will then be physically cut, and the protein complexes extracted from each portion separately. Each extract may then be analysed, such as by [[peptide mass fingerprinting]] or [[de novo peptide sequencing]] after [[in-gel digestion]]. This can provide a great deal of information about the identities of the proteins in a complex.

==Applications==
*Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in [[restriction enzyme|restriction mapping]] of cloned DNA.
*Analysis of [[Polymerase chain reaction|PCR]] products, e.g. in molecular [[Preimplantation genetic diagnosis|genetic diagnosis]] or [[genetic fingerprinting]]
*Separation of restricted genomic DNA prior to [[Southern blot|Southern transfer]], or of RNA prior to [[Northern Blot|Northern transfer]].

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===
{{Main|Gel electrophoresis of nucleic acids}}

[[File: Pcr gel.png|thumb|An agarose gel of a [[Polymerase chain reaction|PCR]] product compared to a DNA ladder.]]

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|title = Molecular Cell Biology|edition = 5th|publisher = WH Freeman: New York, NY|year = 2004|url = https://archive.org/details/molecularcellbio00harv|isbn = 978-0-7167-4366-8|author2 = Berk A|author3 = Matsudaira P|url-access = registration}}</ref>

Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their size or, for cyclic fragments, their [[radius of gyration]]. Circular DNA such as [[plasmid]]s, however, may show multiple bands, the speed of migration may depend on whether it is relaxed or supercoiled.  Single-stranded DNA or RNA tends 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. Characterization through ligand interaction of nucleic acids or fragments may be performed by mobility shift [[affinity electrophoresis]].

Electrophoresis of RNA samples can be used to check for genomic DNA contamination and also for RNA degradation. RNA from eukaryotic organisms shows distinct bands of 28s and 18s rRNA, the 28s band being approximately twice as intense as the 18s band. Degraded RNA has less sharply defined bands, has a smeared appearance, and the intensity ratio is less than 2:1.

===Proteins===
{{Main|Gel electrophoresis of proteins}}

[[File:SDSPAGE.png|thumb|right|'''SDS-PAGE [[autoradiography]]''' – The indicated proteins are present in different concentrations in the two samples.]]
[[Protein]]s, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacrylamide gel at similar rates, or 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]] (SDS) 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 their size and not their 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 preparative native gel electrophoresis ([[QPNC-PAGE]]), or by [[2-D electrophoresis]].

Characterization through ligand interaction may be performed by [[electroblotting]] or by [[affinity electrophoresis]] in agarose or by [[capillary electrophoresis]] as for estimation of [[binding constant]]s and determination of structural features like [[glycan]] content through [[lectin]] binding.

===Nanoparticles===

A novel application for gel electrophoresis is the separation or characterization of metal or metal oxide nanoparticles (e.g. Au, Ag, ZnO, SiO2) regarding the size, shape, or surface chemistry of the nanoparticles.<ref>{{cite journal | url=https://pubs.acs.org/doi/full/10.1021/nl071615y | doi=10.1021/nl071615y | title=Separation of Nanoparticles by Gel Electrophoresis According to Size and Shape | year=2007 | last1=Hanauer | first1=Matthias | last2=Pierrat | first2=Sebastien | last3=Zins | first3=Inga | last4=Lotz | first4=Alexander | last5=Sönnichsen | first5=Carsten | journal=Nano Letters | volume=7 | issue=9 | pages=2881–2885 | pmid=17718532 | bibcode=2007NanoL...7.2881H }}</ref> The scope is to obtain a more homogeneous sample (e.g. narrower particle size distribution), which then can be used in further products/processes (e.g. self-assembly processes). For the separation of nanoparticles within a gel, the key parameter is the ratio of the particle size to the mesh size, whereby two migration mechanisms were identified: the unrestricted mechanism, where the particle size << mesh size, and the restricted mechanism, where particle size is similar to mesh size.<ref name="Barasinski2020">{{cite journal | last1=Barasinski | first1=Matthäus | last2=Garnweitner | first2=Georg | title=Restricted and Unrestricted Migration Mechanisms of Silica Nanoparticles in Agarose Gels and Their Utilization for the Separation of Binary Mixtures | journal=The Journal of Physical Chemistry C | publisher=American Chemical Society (ACS) | volume=124 | issue=9 | date=2020-02-12 | issn=1932-7447 | doi=10.1021/acs.jpcc.9b10644 | pages=5157–5166| s2cid=213566317 }}</ref>

==History==
*1930s – first reports of the use of [[sucrose]] for gel electrophoresis; [[moving-boundary electrophoresis]] ([[Arne Tiselius|Tiselius]])
*1950 – introduction of "zone electrophoresis" (Tiselius); paper electrophoresis
*1955 – introduction of [[starch]] gels, mediocre separation ([[Oliver Smithies|Smithies]])<ref name="pmid13276348"/>
*1959 – introduction of [[acrylamide]] gels; discontinuous electrophoresis ([[Discontinuous electrophoresis|Ornstein and Davis]]); accurate control of parameters such as pore size and stability; and ([[Polyacrylamide|Raymond and Weintraub]])
*1965 – introduction of free-flow electrophoresis ([[free-flow electrophoresis|Hannig]]) 
*1966 – first use of [[agar]] gels<ref name="pmid4287545">{{cite journal | author=Thorne HV | title=Electrophoretic separation of polyoma virus DNA from host cell DNA. | journal=Virology | year=1966 | volume=29 | issue=2 | pages=234–9 | pmid=4287545 | doi=10.1016/0042-6822(66)90029-8 | pmc= | url=https://pubmed.ncbi.nlm.nih.gov/4287545 | access-date=23 March 2022 | archive-date=11 June 2022 | archive-url=https://web.archive.org/web/20220611043441/https://www.sciencedirect.com/science/article/abs/pii/0042682266900298?via%3Dihub | url-status=live }}</ref>
*1969 – introduction of [[Denaturation (biochemistry)|denaturing]] agents especially [[Sodium dodecyl sulfate|SDS]] separation of [[protein]] subunit ([[Klaus Weber|Weber]] and [[Mary Osborn|Osborn]])<ref name="pmid5806584">{{cite journal| author1=Weber K| author2=Osborn M| title=The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. | journal=J Biol Chem | year= 1969 | volume= 244 | issue= 16 | pages= 4406–12 | pmid=5806584 | doi= 10.1016/S0021-9258(18)94333-4| pmc= | doi-access=free }}</ref>
*1970 – [[Ulrich K. Laemmli|Lämmli]] separated 28 components of [[T4 phage]] using a stacking gel and SDS
*1972 – agarose gels with ethidium bromide stain<ref name="pmid5063906">{{cite journal | author1=Aaij C | author2=Borst P | title=The gel electrophoresis of DNA. | journal=Biochim Biophys Acta | year=1972 | volume=269 | issue=2 | pages=192–200 | pmid=5063906 | doi=10.1016/0005-2787(72)90426-1 | pmc= | url=https://pubmed.ncbi.nlm.nih.gov/5063906 | access-date=23 March 2022 | archive-date=11 June 2022 | archive-url=https://web.archive.org/web/20220611043527/https://www.sciencedirect.com/science/article/abs/pii/0005278772904261?via%3Dihub | url-status=live }}</ref>
*1975 – 2-dimensional gels ([[Patrick H. O'Farrell|O’Farrell]]); [[isoelectric focusing]], then SDS gel electrophoresis
*1977 – [[DNA sequencing|sequencing]] gels ([[Frederick Sanger|Sanger]])
*1981 – introduction of capillary electrophoresis [[capillary electrophoresis|(Jorgenson and Lukacs)]]
*1984 – pulsed-field gel electrophoresis enables separation of large DNA molecules ([[pulsed-field gel electrophoresis|Schwartz]] and [[Charles Cantor|Cantor]])
*2004 – introduction of a standardized [[QPNC-PAGE#Gel properties and polymerization time|polymerization time]] for acrylamide [[Solution (chemistry)|solutions]] to optimize [[QPNC-PAGE#Gel properties and polymerization time|gel properties]], in particular [[Chemical stability|gel stability]] ([[QPNC-PAGE|Kastenholz]])<ref>{{cite book |author=Michov, B. |year=2022 |title=Electrophoresis Fundamentals: Essential Theory and Practice |url=https://www.degruyter.com/document/doi/10.1515/9783110761641/html |url-access= |publisher=De Gruyter, ISBN 9783110761627|doi=10.1515/9783110761641 |isbn=9783110761641 }}</ref>

A 1959 book on electrophoresis by Milan Bier cites references from the 1800s.<ref name="Bier1959">{{cite book | last=Bier | first=Milan | title=Electrophoresis: theory, methods, and applications | publisher=Academic Press | year=1959 | oclc=1175404 | page=225}}</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==
{{div col|colwidth=30em}}
* [[History of electrophoresis]]
* [[Electrophoretic mobility shift assay]]
* [[Gel extraction]]
* [[Isoelectric focusing]]
* [[Pulsed field gel electrophoresis]]
* [[Nonlinear frictiophoresis]]
* [[Two-dimensional gel electrophoresis]]
* [[SDD-AGE]]
* [[QPNC-PAGE]]
* [[Zymography]]
* [[Fast parallel proteolysis]]<ref name="Minde2012">{{cite journal | last1=Minde | first1=David P. | last2=Maurice | first2=Madelon M. | last3=Rüdiger | first3=Stefan G. D. | editor-last=Uversky | editor-first=Vladimir N. | title=Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp | journal=PLOS ONE | publisher=Public Library of Science (PLoS) | volume=7 | issue=10 | date=2012-10-03 | issn=1932-6203 | pmid=23056252 | pmc=3463568 | doi=10.1371/journal.pone.0046147 | page=e46147| bibcode=2012PLoSO...746147M | doi-access=free }}</ref>
* [[Free-flow electrophoresis]]
{{div col end}}

==References==
{{Reflist}}

==External links==
{{Commons category}}
* [https://learn.genetics.utah.edu/content/labs/gel/ Biotechniques Laboratory electrophoresis demonstration], from the University of Utah's Genetic Science Learning Center
* [https://archive.today/20121217205427/http://www3.interscience.wiley.com/cgi-bin/abstract/113343976/ABSTRACT Discontinuous native protein gel electrophoresis]
* [https://web.archive.org/web/20090210181129/http://maradydd.livejournal.com/417631.html Drinking straw electrophoresis]
*[https://web.archive.org/web/20110927050058/http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/index.html How to run a DNA or RNA gel]
*[https://web.archive.org/web/20110927050110/http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html Animation of gel analysis of DNA restriction]
*[https://web.archive.org/web/20151020012948/http://web.mit.edu:80/7.02/virtual_lab/RDM/RDM1virtuallab.html Step by step photos of running a gel and extracting DNA]
*[[wikiversity:Agarose gel electrophoresis|A typical method from wikiversity]]

{{Protein methods}}
{{electrophoresis}}

{{DEFAULTSORT:Gel Electrophoresis}}
[[Category:Protein methods]]
[[Category:Molecular biology]]
[[Category:Laboratory techniques]]
[[Category:Electrophoresis]]
[[Category:Polymerase chain reaction]]
[[Category:Gels|electrophoresis]]