Agarose
Agarose is a
Agarose is frequently used in
Structure
Agarose is a linear polymer with a molecular weight of about 120,000, consisting of alternating D-
Each agarose chain contains ~800 molecules of galactose, and the agarose polymer chains form helical fibers that aggregate into supercoiled structure with a radius of 20-30 nanometer (nm).[6] The fibers are quasi-rigid, and have a wide range of length depending on the agarose concentration.[7] When solidified, the fibers form a three-dimensional mesh of channels of diameter ranging from 50 nm to >200 nm depending on the concentration of agarose used - higher concentrations yield lower average pore diameters. The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state.
Properties
Agarose is available as a white powder which dissolves in near-boiling water, and forms a gel when it cools. Agarose exhibits the phenomenon of thermal hysteresis in its liquid-to-gel transition, i.e. it gels and melts at different temperatures. The gelling and melting temperatures vary depending on the type of agarose. Standard agaroses derived from Gelidium has a gelling temperature of 34–38 °C (93–100 °F) and a melting temperature of 90–95 °C (194–203 °F), while those derived from Gracilaria, due to its higher methoxy substituents, has a gelling temperature of 40–52 °C (104–126 °F) and melting temperature of 85–90 °C (185–194 °F).[8] The melting and gelling temperatures may be dependent on the concentration of the gel, particularly at low gel concentration of less than 1%. The gelling and melting temperatures are therefore given at a specified agarose concentration.
Natural agarose contains uncharged methyl groups and the extent of methylation is directly proportional to the gelling temperature. Synthetic methylation however have the reverse effect, whereby increased methylation lowers the gelling temperature.[9] A variety of chemically modified agaroses with different melting and gelling temperatures are available through chemical modifications.
The agarose in the gel forms a meshwork that contains pores, and the size of the pores depends on the concentration of agarose added. On standing, the agarose gels are prone to syneresis (extrusion of water through the gel surface), but the process is slow enough to not interfere with the use of the gel.[10][11]
Agarose gel can have high gel strength at low concentration, making it suitable as an anti-convection medium for
Low melting and gelling temperature agaroses
The melting and gelling temperatures of agarose can be modified by chemical modifications, most commonly by hydroxyethylation, which reduces the number of intrastrand hydrogen bonds, resulting in lower melting and setting temperatures compared to standard agaroses.[15] The exact temperature is determined by the degree of substitution, and many available low-melting-point (LMP) agaroses can remain fluid at 30–35 °C (86–95 °F) range. This property allows enzymatic manipulations to be carried out directly after the DNA gel electrophoresis by adding slices of melted gel containing DNA fragment of interest to a reaction mixture. The LMP agarose contains fewer of the sulphates that can affect some enzymatic reactions, and is therefore preferably used for some applications.
Hydroxyethylated agarose also has a smaller pore size (~90 nm) than standard agaroses.[16] Hydroxyethylation may reduce the pore size by reducing the packing density of the agarose bundles, therefore LMP gel can also have an effect on the time and separation during electrophoresis.[17] Ultra-low melting or gelling temperature agaroses may gel only at 8–15 °C (46–59 °F).
Applications
Agarose is a preferred matrix for work with proteins and nucleic acids 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 biomolecules. Agarose is most commonly used as the medium for analytical scale 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 immunocomplexes.
Agarose gel electrophoresis
Agarose gel electrophoresis is the routine method for resolving
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.[18] 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.
Protein purification
Agarose gel matrix is often used for
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 denaturants such as 8M urea or 6M guanidine HCl.[21] 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.[22] 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,[23] Caenorhabditis elegans,[24] 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
See also
References
- ^ PMID 313856.
- .
- ^ Agar Archived October 16, 2007, at the Wayback Machine at lsbu.ac.uk Water Structure and Science
- ^ "Agar". Food and Agricultural Organization of the United Nations.
- ^ Armisen R, Galatas F. "Chapter 1 - Production, Properties and Uses of Agar". Fao.org.
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- ISBN 978-0824759223.
- ^ Workshop on Marine Algae Biotechnology: Summary Report. National Academy Press. 1986. p. 25.
- ^ a b "Appendix B: Agarose Physical Chemistry" (PDF). Lonza Group. Archived (PDF) from the original on 2022-10-09.
- ISBN 978-0-7514-0421-0.
- ISBN 978-1566760041.
- ^ S2CID 97819634.
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- ^ ISBN 978-0879691363.
- ISBN 978-0716714446.
- ^ "Overview of Affinity Purification". Thermo Scientific.
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