Editing Gel electrophoresis (section)

==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>