Editing Agarose (section)

==Applications==
[[File:AgarosegelUV.jpg|thumb|An agarose gel with bands of DNA stained with ethidium bromide and visualized under [[Ultraviolet|UV light]] on a UV Transilluminator.]]
Agarose is a preferred matrix for work with [[protein]]s and [[nucleic acid]]s as it has a broad range of physical, chemical and thermal stability, and its lower degree of chemical complexity also makes it less likely to interact with [[biomolecule]]s. Agarose is most commonly used as the medium for analytical scale [[Electrophoresis|electrophoretic]] separation in [[agarose gel electrophoresis]]. Gels made from purified agarose have a relatively large pore size, making them useful for separation of large molecules, such as proteins and protein complexes >200 kilodaltons, as well as DNA fragments >100 basepairs. Agarose is also used widely for a number of other applications, for example [[immunodiffusion]] and [[immunoelectrophoresis]], as the agarose fibers can function as anchor for [[Immune complex|immunocomplexes]].

=== Agarose gel electrophoresis ===
{{Main|Agarose gel electrophoresis}}

Agarose gel electrophoresis is the routine method for resolving [[DNA]] in the laboratory. Agarose gels have lower resolving power for DNA than [[acrylamide]] gels, but they have greater range of separation, and are therefore usually used for DNA fragments with lengths of 50–20,000 bp ([[base pair]]s), although resolution of over 6 Mb is possible with [[pulsed field gel electrophoresis]] (PFGE).<ref name="maniatis1">{{cite book |title=Molecular Cloning - A Laboratory Manual | vauthors = Maniatis T, Fritsch EF, Sambrook J |volume=1 |chapter=Chapter 5, protocol 1 |year=1982 |page=5.2–5.3 |isbn=978-0879691363 }}</ref> It can also be used to separate large protein molecules, and it is the preferred matrix for the gel electrophoresis of particles with effective [[Radius|radii]] larger than 5-10&nbsp;nm.<ref name="serwer">{{cite journal |title=Agarose gels: Properties and use for electrophoresis | vauthors = Serwer P |journal=Electrophoresis |volume = 4 |issue =6 |pages =375–382 |year= 1983 |doi=10.1002/elps.1150040602|s2cid =97819634 }}</ref>

The pore size of the gel affects the size of the DNA that can be sieved. The lower the concentration of the gel, the larger the pore size, and the larger the DNA that can be sieved. However low-concentration gels (0.1 - 0.2%) are fragile and therefore hard to handle, and the electrophoresis of large DNA molecules can take several days. The limit of resolution for standard agarose gel electrophoresis is around 750 kb.<ref name="maniatis1" /> This limit can be overcome by PFGE, where alternating orthogonal electric fields are applied to the gel. The DNA fragments reorientate themselves when the applied field switches direction, but larger molecules of DNA take longer to realign themselves when the electric field is altered, while for smaller ones it is quicker, and the DNA can therefore be fractionated according to size.

Agarose gels are cast in a mold, and when set, usually run horizontally submerged in a buffer solution. [[TAE buffer|Tris-acetate-EDTA]] and [[TBE buffer|Tris-Borate-EDTA]] buffers are commonly used, but other buffers such as Tris-phosphate, barbituric acid-sodium barbiturate or Tris-[[Barbital|barbiturate]] buffers may be used in other applications.<ref name="jeppson"/> The DNA is normally visualized by staining with [[ethidium bromide]] and then viewed under a [[UV light]], but other methods of staining are available, such as [[SYBR Green I|SYBR Green]], [[GelRed]], [[methylene blue]], and [[crystal violet]]. If the separated DNA fragments are needed for further downstream experiment, they can be cut out from the gel in slices for further manipulation.

=== Protein purification ===
[[File:Size_Exclusion_Chromatography_Apparatus.jpg|thumb|Agarose-based gel filtration columns used for protein purification on an AKTA [[Fast protein liquid chromatography|FPLC]] machine.]]

Agarose gel matrix is often used for [[protein purification]], for example, in column-based preparative scale separation as in [[Size exclusion chromatography|gel filtration chromatography]], [[affinity chromatography]] and [[ion exchange chromatography]]. It is however not used as a continuous gel, rather it is formed into porous beads or resins of varying fineness.<ref name=freifelder>{{cite book |title=Physical Biochemistry: Applications to Biochemistry and Molecular Biology | vauthors = Freifelder D |edition=2nd |year=1982 |publisher=WH Freeman |page=240 |isbn= 978-0716714446}}</ref> The beads are highly porous so that protein may flow freely through the beads. These agarose-based beads are generally soft and easily crushed, so they should be used under gravity-flow, low-speed centrifugation, or low-pressure procedures.<ref>{{cite web |url=http://www.piercenet.com/method/overview-affinity-purification |title=Overview of Affinity Purification |work=Thermo Scientific }}</ref> The strength of the resins can be improved by increased cross-linking and chemical hardening of the agarose resins, however such changes may also result in a lower binding capacity for protein in some separation procedures such as [[affinity chromatography]].

Agarose is a useful material for chromatography because it does not absorb biomolecules to any significant extent, has good flow properties, and can tolerate extremes of [[pH]] and [[ionic strength]] as well as high concentration of [[Denaturation (biochemistry)|denaturant]]s such as 8M [[urea]] or 6M [[Guanidinium chloride|guanidine HCl]].<ref>{{cite book |title=Physical Biochemistry: Applications to Biochemistry and Molecular Biology | vauthors = Freifelder D |edition=2nd |year=1982 |publisher=WH Freeman |page= 258 |isbn= 978-0716714446}}</ref> Examples of agarose-based matrix for gel filtration chromatography are [[Sepharose]] and WorkBeads 40 SEC (cross-linked beaded agarose), ''Praesto'' and [[Superose]] (highly cross-linked beaded agaroses), and Superdex ([[dextran]] covalently linked to agarose).

For affinity chromatography, beaded agarose is the most commonly used matrix resin for the attachment of the ligands that bind protein.<ref>{{cite book |title=Encyclopedia of Biological Chemistry |volume= 1 |page=52 | vauthors = Cuatrecasas P, Wilchek M | veditors = Lennarz WJ, Lane MD |publisher=Academic Press |isbn= 9780124437104 |year=2004}}</ref> The ligands are linked covalently through a spacer to activated hydroxyl groups of agarose bead polymer. Proteins of interest can then be selectively bound to the ligands to separate them from other proteins, after which it can be eluted. The agarose beads used are typically of 4% and 6% densities with a high binding capacity for protein.

===Solid culture media===
Agarose plate may sometimes be used instead of agar for culturing organisms as agar may contain impurities that can affect the growth of the organism or some downstream procedures such as [[polymerase chain reaction]] (PCR). Agarose is also harder than agar and may therefore be preferable where greater gel strength is necessary, and its lower gelling temperature may prevent causing [[thermal shock]] to the organism when the cells are suspended in liquid before gelling. It may be used for the culture of strict autotrophic bacteria, plant [[protoplast]],<ref>{{cite book |url=https://books.google.com/books?id=vjI5KXFZlcoC&pg=PA16 |title=In Vitro Culture of Trees| vauthors = Bonga JM, von Aderkas P |page=16 |year= 1992 |publisher=Springer |isbn=978-0792315407 }}</ref> ''[[Caenorhabditis elegans]]'',<ref>{{cite book |url=https://books.google.com/books?id=bxZeffKo4-8C&pg=PA95 |title=Integrated Genomics: A Discovery-Based Laboratory Course| vauthors = Caldwell GA, Williams SN, Caldwell KA |pages=94–95 |year= 2006 |publisher=Wiley |isbn= 978-0470095027 }}</ref> other organisms and various cell lines.

=== Motility assays ===
Agarose is sometimes used instead of agar to measure microorganism motility and mobility. Motile species will be able to migrate, albeit slowly, throughout the porous gel and infiltration rates can then be visualized. The gel's porosity is directly related to the concentration of agar or agarose in the medium, so different concentration gels may be used to assess a cell's [[Flagellar motility|swimming]], [[Swarming motility|swarming]], [[gliding motility|gliding]] and twitching motility. Under-agarose cell migration assay may be used to measure [[chemotaxis]] and chemokinesis. A layer of agarose gel is placed between a cell population and a [[chemoattractant]]. As a concentration gradient develops from the [[diffusion]] of the chemoattractant into the gel, various cell populations requiring different stimulation levels to migrate can then be visualized over time using microphotography as they tunnel upward through the gel against gravity along the gradient.