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Agarose
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used for gel electrophoresis
Agarose is a heteropolysaccharide, generally extracted from certain red algae.[1] It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose.[2][3] Agarose is one of the two principal components of agar, and is purified from agar by removing agar's other component, agaropectin.[4]
Agarose is frequently used in molecular biology for the separation of large molecules, especially DNA, by electrophoresis. Slabs of agarose gels (usually 0.7 - 2%) for electrophoresis are readily prepared by pouring the warm, liquid solution into a mold. A wide range of different agaroses of varying molecular weights and properties are commercially available for this purpose. Agarose may also be formed into beads and used in a number of chromatographic methods for protein purification.
Structure
[edit]
Agarose is a linear polymer with a molecular weight of about 120,000, consisting of alternating D-galactose and 3,6-anhydro-L-galactopyranose linked by α-(1→3) and β-(1→4) glycosidic bonds. The 3,6-anhydro-L-galactopyranose is an L-galactose with an anhydro bridge between the 3 and 6 positions, although some L-galactose units in the polymer may not contain the bridge. Some D-galactose and L-galactose units can be methylated, and pyruvate and sulfate are also found in small quantities.[5]
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
[edit]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 gel electrophoresis. Agarose gels as dilute as 0.15% can form slabs for gel electrophoresis.[12] The agarose polymer contains charged groups, in particular pyruvate and sulfate.[9] These negatively charged groups can slow down the movement of DNA molecules in a process called electroendosmosis (EEO).
Low EEO (LE) agarose is therefore generally preferred for use in agarose gel electrophoresis of nucleic acids. Zero EEO agaroses are also available but these may be undesirable for some applications as they may be made by adding positively charged groups that can affect subsequent enzyme reactions.[13] Electroendosmosis is a reason agarose is used preferentially over agar as agaropectin in agar contains a significant amount of negatively charged sulphate and carboxyl groups. The removal of agaropectin in agarose substantially reduces the EEO, as well as reducing the non-specific adsorption of biomolecules to the gel matrix. However, for some applications such as the electrophoresis of serum protein, a high EEO may be desirable, and agaropectin may be added in the gel used.[14]
LE agarose is said to be better for preparative electrophoresis, i.e. when DNA needs to be extracted from an agarose gel.[15]
Low melting and gelling temperature agaroses
[edit]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.[16] 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.[17] 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.[18] Ultra-low melting or gelling temperature agaroses may gel only at 8–15 °C (46–59 °F).
Applications
[edit]
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
[edit]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 pairs), although resolution of over 6 Mb is possible with pulsed field gel electrophoresis (PFGE).[19] It can also be used to separate large protein molecules, and it is the preferred matrix for the gel electrophoresis of particles with effective radii larger than 5-10 nm.[12]
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.[19] 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. Tris-acetate-EDTA and Tris-Borate-EDTA buffers are commonly used, but other buffers such as Tris-phosphate, barbituric acid-sodium barbiturate or Tris-barbiturate buffers may be used in other applications.[1] 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, 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
[edit]
Agarose gel matrix is often used for protein purification, for example, in column-based preparative scale separation as in 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.[20] 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.[21] 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 denaturants such as 8M urea or 6M guanidine HCl.[22] 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.[23] 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
[edit]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,[24] Caenorhabditis elegans,[25] other organisms and various cell lines.
Motility assays
[edit]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 swimming, swarming, 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.
See also
[edit]References
[edit]- ^ a b Jeppsson JO, Laurell CB, Franzén B (April 1979). "Agarose gel electrophoresis". Clinical Chemistry. 25 (4): 629–38. doi:10.1093/clinchem/25.4.629. PMID 313856.
- ^ Akari C (1956). "Structure of the agarose component of agar-agar". Bulletin of the Chemical Society of Japan. 29 (4): 543–544. doi:10.1246/bcsj.29.543.
- ^ 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.
- ^ Maniatis T, Fritsch EF, Sambrook J (1982). "Chapter 5, protocol 1". Molecular Cloning - A Laboratory Manual. Vol. 1. p. 5.4. ISBN 978-0879691363.
- ^ Stephen AM, Phillips GO, eds. (2006). Food Polysaccharides and Their Applications. CRC Press. p. 226. 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.
- ^ Hill SE, Ledward DA, Mitchell JR, eds. (1998). Functional Properties of Food Macromolecules. Springer. p. 149. ISBN 978-0-7514-0421-0.
- ^ Park H, Park K, Shalaby WS (1993). Biodegradable Hydrogels for Drug Delivery. CRC Press. p. 102. ISBN 978-1566760041.
- ^ a b Serwer P (1983). "Agarose gels: Properties and use for electrophoresis". Electrophoresis. 4 (6): 375–382. doi:10.1002/elps.1150040602. S2CID 97819634.
- ^ Sambrook J, Russell D. "Chapter 5, protocol 1". Molecular Cloning - A Laboratory Manual. Vol. 1 (3rd ed.). p. 5.7. ISBN 978-0-87969-577-4.
- ^ Keren D (26 September 2003). Protein Electrophoresis in Clinical Diagnosis. CRC Press. pp. 7–8. ISBN 978-0340812136.
- ^ Martin, Katherine. "Agarose LE vs. Agarose – What's the Difference?". Gold Biotechnology. Retrieved 2024-09-19.
- ^ Maniatis T, Fritsch EF, Sambrook J. "Chapter 5, protocol 6". Molecular Cloning - A Laboratory Manual. Vol. 1. p. 5.29. ISBN 978-0879695774.
- ^ Griess, Gary A.; Moreno, Elena T.; Easom, Richard A.; Serwer, Philip (1989). "The sieving of spheres during agarose gel electrophoresis: Quantitation and modeling". Biopolymers. 28 (8). Wiley: 1475–1484. doi:10.1002/bip.360280811. ISSN 0006-3525.
- ^ Lee PY, Costumbrado J, Hsu CY, Kim YH (April 2012). "Agarose gel electrophoresis for the separation of DNA fragments". Journal of Visualized Experiments. 62 (62): 3923. doi:10.3791/3923. PMC 4846332. PMID 22546956.
- ^ a b Maniatis T, Fritsch EF, Sambrook J (1982). "Chapter 5, protocol 1". Molecular Cloning - A Laboratory Manual. Vol. 1. p. 5.2–5.3. ISBN 978-0879691363.
- ^ Freifelder D (1982). Physical Biochemistry: Applications to Biochemistry and Molecular Biology (2nd ed.). WH Freeman. p. 240. ISBN 978-0716714446.
- ^ "Overview of Affinity Purification". Thermo Scientific. Archived from the original on 2013-10-17. Retrieved 2013-10-17.
- ^ Freifelder D (1982). Physical Biochemistry: Applications to Biochemistry and Molecular Biology (2nd ed.). WH Freeman. p. 258. ISBN 978-0716714446.
- ^ Cuatrecasas P, Wilchek M (2004). Lennarz WJ, Lane MD (eds.). Encyclopedia of Biological Chemistry. Vol. 1. Academic Press. p. 52. ISBN 9780124437104.
- ^ Bonga JM, von Aderkas P (1992). In Vitro Culture of Trees. Springer. p. 16. ISBN 978-0792315407.
- ^ Caldwell GA, Williams SN, Caldwell KA (2006). Integrated Genomics: A Discovery-Based Laboratory Course. Wiley. pp. 94–95. ISBN 978-0470095027.
Agarose
View on GrokipediaDefinition and Sources
Natural Occurrence
Agarose is a principal polysaccharide component of agar, a hydrocolloid primarily extracted from species of red algae (Rhodophyta) belonging to the orders Gelidiales and Gracilariales.[4] The most commercially significant sources include genera such as Gelidium, Gracilaria, and Pterocladia, where agarose constitutes approximately 60-70% of the total agar fraction.[5] These algae synthesize agarose as part of their intercellular matrix and cell wall matrix, enabling its accumulation in substantial quantities within the algal biomass.[6] Red algae producing agarose are widely distributed in marine ecosystems, thriving in coastal intertidal and subtidal zones of temperate and tropical oceans globally. Gelidium species predominate in cooler temperate waters, such as those along the Atlantic coasts of Europe and North America, the Mediterranean Sea, and parts of the Pacific Ocean.[7] In contrast, Gracilaria exhibits a broader range in warmer tropical and subtropical regions, including the Indo-Pacific, Caribbean, and Indian Ocean waters, while Pterocladia is found in similar temperate to subtropical locales, notably the Atlantic islands and New Zealand coasts.[8] This distribution reflects adaptations to varying salinity, light, and temperature conditions in benthic marine habitats.[9] In algal physiology, agarose contributes to the structural integrity of cell walls, forming a gel-like network that provides mechanical support and resilience against environmental stresses, including desiccation during low tides and fluctuations in osmotic pressure.[5] This role aids in maintaining cellular turgor and preventing collapse under dehydration or salinity shifts, complementing other osmoregulatory mechanisms like the accumulation of low-molecular-weight carbohydrates.[10] The agarose content varies significantly across species and environmental conditions; for instance, in Gelidium, it can comprise 20-50% of the dry weight, depending on factors such as growth stage and habitat, whereas Gracilaria typically yields lower proportions, around 10-40%.[11][12]Extraction and Purification
The extraction of agarose begins with the harvesting and cleaning of red algae, primarily species such as Gelidium and Gracilaria, which are washed to remove sand, salt, and debris.[13] The cleaned algae undergo an alkaline pretreatment, typically involving immersion in 2-5% sodium hydroxide (NaOH) solution at 85-95°C for 0.5-1 hour, which modifies the structure of the agar by reducing sulfate content to enhance gelling properties, while the subsequent steps separate agarose from the charged agaropectin.[13][2] Following this, the pretreated algae are subjected to hot water extraction, often at 90-100°C under pressure for 1-3 hours, to solubilize the agarose-rich fraction into an aqueous solution. Purification commences with coarse filtration of the hot extract to eliminate algal residues and insoluble materials, followed by finer filtration using aids like diatomaceous earth to clarify the solution.[13] To separate agarose from agaropectin, potassium chloride (KCl) is added to the cooled extract, selectively precipitating the charged agaropectin while leaving agarose in the supernatant; the supernatant is then treated with ethanol (typically 50-70% concentration) to precipitate the agarose as a gel-like mass.[14] The precipitated agarose is filtered, washed with ethanol or water to remove impurities, and dried under vacuum or at low temperatures (40-60°C) to produce a fine, white powder suitable for commercial use.[2][15] Yields of agarose from dry algal biomass typically range from 10-30%, varying by species—for instance, Gracilaria cervicornis yields 11-20%—and are influenced by seasonal factors such as nutrient availability and temperature, with higher outputs often during dry seasons due to concentrated biomass.[16] Processing conditions, including NaOH concentration and extraction temperature, also affect yields, with optimized alkaline treatments increasing recovery by up to 20%.[17] Modern extraction processes incorporate environmental considerations to promote sustainability, such as regulated harvesting quotas and cultivation of algal farms to prevent overexploitation of natural beds, which has historically led to depletion in regions like the Mediterranean.[18] Efforts also include reducing alkali and solvent usage through greener alternatives like enzymatic pretreatments or ionic liquids, minimizing wastewater pollution and energy consumption in line with life-cycle assessments showing high impacts from traditional methods.[19][20]Chemical Structure
Monomer Composition
Agarose is a linear polysaccharide composed of repeating disaccharide units known as agarobiose.[21] The fundamental monomers of agarobiose are β-D-galactopyranose and 3,6-anhydro-α-L-galactopyranose, which alternate in the polymer chain.[21] These units are connected through specific glycosidic bonds: a β-1,4 linkage joins the β-D-galactopyranose to the 3,6-anhydro-α-L-galactopyranose, while an α-1,3 linkage connects the 3,6-anhydro-α-L-galactopyranose to the subsequent β-D-galactopyranose unit.[21] This arrangement forms the repeating agarobiose motif, represented structurally as: where Galp denotes galactopyranose.[21] Both monomers are neutral sugars, with the 3,6-anhydro bridge in the α-L-galactopyranose unit imparting conformational rigidity to the disaccharide.[21] In contrast to cellulose, which consists exclusively of β-D-glucopyranose monomers linked by uniform β-1,4 glycosidic bonds, agarose employs two distinct galactose-derived units with alternating α and β linkages.[22] Similarly, unlike pectin—a polysaccharide primarily built from α-D-galacturonic acid monomers connected via α-1,4 bonds and featuring acidic carboxyl groups—agarose remains neutral due to its unmodified sugar composition and inclusion of the anhydro bridge.Polymer Characteristics
Agarose is a linear, unbranched polysaccharide polymer composed of repeating agarobiose units, forming long chains with molecular weights typically ranging from 100,000 to 150,000 Da, corresponding to a degree of polymerization of approximately 300 to 500 units.[23][24] These chains are neutral and free of significant branching, which contributes to their uniformity and functionality in various applications. Upon cooling in aqueous solutions, agarose chains form a double-helical conformation, where two polymer strands intertwine, stabilized primarily by hydrogen bonding between hydroxyl groups on adjacent sugar residues.[2] This helical structure promotes interchain associations, facilitating the aggregation observed in gel formation. Purity of agarose is critically assessed through metrics such as the electroendosmosis (EEO) value, which quantifies the presence of charged impurities like sulfate ester groups that introduce negative charges along the polymer backbone.[26] Low EEO values (e.g., <0.16) indicate high purity with minimal sulfate content, reducing unwanted ionic effects during use.[27] Spectroscopic techniques, including nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy, are employed to confirm the uniformity of agarose chains and the absence of contaminants such as agaropectin, a sulfated fraction of agar.[28] For instance, 13C-NMR spectra of purified agarose display distinct signals for the anhydrogalactose and galactose residues without additional peaks characteristic of agaropectin's modified structures, while IR analysis identifies key vibrational bands associated with the neutral polysaccharide backbone.[29]Physical and Chemical Properties
Solubility and Gel Formation
Agarose exhibits limited solubility in water at ambient temperatures, remaining largely insoluble in cold conditions due to its polymeric structure, but it dissolves readily when heated to boiling (approximately 95-100°C), allowing for complete hydration and dispersion in solution.[30][31] This thermal solubility threshold enables the preparation of agarose solutions typically at concentrations of 0.5% to 2% w/v, which are standard for forming gels suitable for various applications. The process requires boiling or heating to ensure uniform dissolution without degradation, as incomplete solubilization can lead to heterogeneous gels.[30] Upon cooling the solubilized agarose solution to around 30–40°C, gelation occurs through a reversible thermoreversible mechanism, where random coil conformations transition into double helices stabilized by intermolecular hydrogen bonds, followed by aggregation of these helices into a three-dimensional network that traps water molecules.[2] This hydrogen bond-mediated assembly forms a porous matrix without covalent cross-links, allowing the gel to melt upon reheating and reform upon recooling, a property central to agarose's utility in dynamic systems.[32] The resulting gel structure is non-toxic and biocompatible, supporting its use in biological contexts. The pore size within the agarose gel matrix is inversely proportional to the polymer concentration, with higher concentrations yielding smaller pores due to denser helix packing. For instance, at a 1% concentration, the median pore size is approximately 100 nm, providing sieving capabilities for macromolecules while permitting diffusion of smaller solutes.[33] This concentration-dependent porosity influences the gel's selectivity, with lower concentrations (e.g., 0.5%) producing larger pores for resolving high-molecular-weight species.[34] Gel strength, quantified as the force required to deform the gel (in g/cm²), is significantly influenced by the sulfate content and chain length of the agarose polymer. Lower sulfate levels enhance gel firmness by reducing electrostatic repulsion between chains, promoting tighter helix aggregation, while longer chain lengths increase the density of interconnecting junctions in the network, thereby elevating overall mechanical rigidity.[35] For high-quality agarose, gel strengths often exceed 1200 g/cm² at 1% concentration.[36][37]Thermal and Rheological Properties
Agarose gels exhibit distinct thermal transitions that are critical for their practical applications. Standard agarose gels typically gel upon cooling to temperatures between 32°C and 40°C, forming a stable network of double helices that trap water molecules. In contrast, these gels melt at significantly higher temperatures, ranging from 85°C to 95°C, due to the energy required to disrupt the hydrogen-bonded structure. This temperature hysteresis—where the gelling point is much lower than the melting point—arises from the cooperative formation and dissociation of the helical aggregates, enabling reversible gel-sol transitions without degradation under repeated cycles.[38][39][40] Rheologically, agarose gels display solid-like viscoelastic behavior, characterized by a storage modulus (G') that exceeds the loss modulus (G'') across a range of frequencies, confirming their elastic dominance over viscous flow. Typical G' values for 1-2% agarose gels fall in the range of 10^4 to 10^5 Pa, reflecting the mechanical strength imparted by the interconnected fibrous network. Under applied shear stress, these gels demonstrate shear-thinning properties, where viscosity decreases with increasing shear rate, facilitating easier handling, injection, or extrusion while allowing rapid recovery of structure upon stress removal.[41][42] The neutral chemical structure of agarose contributes to its high stability against enzymatic degradation, distinguishing it from charged polysaccharides like alginate or carrageenan that are more susceptible to glycosidases and other hydrolases. This inertness stems from the absence of charged groups, reducing electrostatic interactions with enzymes and preserving gel integrity in biological environments. As a result, agarose maintains structural stability during prolonged exposure to common proteases and nucleases used in molecular biology protocols.[21][2]Modifications and Variants
Low-Melting and Gelling Temperature Types
Low-melting and low-gelling temperature types of agarose were developed in the 1980s to support applications involving heat-sensitive materials, such as the recovery of intact biomolecules. These variants are achieved through chemical modifications, including hydroxyethylation or methylation, which reduce the stability of the agarose double-helix structure responsible for gel formation.[43][28] Key properties of these agaroses include gelling temperatures of 24–30°C and melting temperatures around 65°C, enabling the use of milder conditions than standard agarose. This facilitates the recovery of entrapped substances, like DNA, by simple heating to 37°C, a temperature compatible with biological activity.[44][45] Such variants prove useful for DNA cloning, where fragments can be excised and recovered without denaturation, and for culturing thermolabile cells, allowing embedding at near-physiological temperatures.[46][47] A notable trade-off is the production of softer gels with reduced resolution for small DNA fragments relative to standard agarose, due to altered polymer interactions.[48]Specialized Agaroses
High-resolution agarose is designed for superior separation of small nucleic acid fragments, achieved primarily through higher agarose concentrations that reduce pore sizes to approximately 50–200 nm, enabling resolution of DNA molecules as small as 50 bp. These gels exhibit low electroendosmosis due to minimized sulfate content (typically <0.11%), which reduces counterflow and band distortion during electrophoresis.[49][27] Affinity agarose incorporates covalently attached ligands, such as antibodies or lectins, to the agarose matrix, facilitating specific biospecific interactions for the purification of target proteins or glycoproteins. For instance, lectin-functionalized agarose beads selectively bind glycosylated molecules through carbohydrate recognition, allowing efficient isolation in chromatographic setups. This modification leverages the inert, porous nature of agarose while introducing high selectivity, with binding affinities often exceeding 10^6 M^{-1} for complementary interactions.[50][51] Cross-linked agarose enhances mechanical stability by introducing chemical bridges, commonly via divinyl sulfone activation, resulting in beaded matrices that resist compression under high flow rates in chromatography (up to 300 cm/h without deformation). This cross-linking increases rigidity while preserving porosity, making it ideal for large-scale purification processes where standard agarose would collapse. The degree of cross-linking can be tuned to balance stability and diffusion rates, with highly cross-linked variants showing gel strengths over 500 g/cm².[52][53] Post-2020 advancements in agarose engineering have focused on chemical and physical modifications to create customizable variants with tailored properties, such as altered swelling or bioactivity, for specialized biomedical applications like drug delivery and tissue scaffolds. Recent research as of 2025 has emphasized the preparation, cross-linking, and activation of agarose microspheres to enhance their biomedical utility.[54][55]Applications
Gel Electrophoresis
Agarose gel electrophoresis is a widely used technique for separating DNA and RNA fragments by size through the application of an electric field across a porous gel matrix. The negatively charged nucleic acid molecules migrate toward the positive electrode (anode), with separation occurring due to the sieving effect of the agarose network, where smaller fragments travel faster through the pores while larger ones are impeded. This size-based fractionation follows principles such as the biased reptation model, in which the leading end of the DNA molecule advances and pulls the rest through the gel, influenced by factors like fragment conformation, agarose concentration, applied voltage, and buffer conditions.[56][23][57] The protocol begins with gel preparation, where agarose powder is weighed to achieve a concentration of 0.5–2% w/v (typically 0.7–1% for general use) and dissolved in an electrophoresis buffer such as TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). The mixture is heated until fully dissolved, cooled to approximately 50–60°C to prevent denaturation, and optionally supplemented with a DNA-intercalating dye like ethidium bromide (0.5 μg/mL); it is then poured into a horizontal casting tray equipped with a comb to create sample wells and allowed to solidify at room temperature for 20–30 minutes. Samples are prepared by mixing DNA or RNA with a loading dye containing glycerol for density and tracking dyes like bromophenol blue, then loaded into the wells of the submerged gel within an electrophoresis chamber filled with the same running buffer. Electrophoresis is conducted at 5–10 V/cm (often 80–150 V total) for 30–90 minutes, with progress monitored by the migration of the dye front to about 75–80% of the gel length.[30][56][23] This method offers effective resolution for linear DNA fragments ranging from 100 bp to 25 kb, with optimal separation achieved by adjusting gel concentration—lower percentages (e.g., 0.5%) for larger fragments and higher (e.g., 2%) for smaller ones—though fragments beyond 25 kb require specialized techniques like pulsed-field electrophoresis. Post-run, the gel is visualized by staining with fluorescent dyes such as ethidium bromide or safer alternatives like SYBR Safe, which intercalate with DNA and emit light under UV illumination, allowing band detection via gel documentation systems.[56][57][23] Compared to polyacrylamide gels, agarose offers advantages including simpler preparation without toxic polymerization chemicals, greater mechanical strength for handling low-concentration gels suitable for large DNA, and non-toxicity, making it preferable for routine separation of fragments above 100 bp while allowing easier recovery of DNA by melting the gel at low temperatures.[56][23][30]Chromatographic and Purification Techniques
Agarose serves as a versatile matrix in various chromatographic techniques for the purification of proteins and other biomolecules, primarily through its use in beaded forms that facilitate column-based separations. Key types include size-exclusion chromatography (SEC), where Sepharose beads act as porous gels to separate molecules by hydrodynamic volume; ion-exchange chromatography (IEC), employing charged agarose matrices like DEAE-Sepharose or CM-Sepharose to separate based on net charge; and affinity chromatography, which utilizes agarose supports functionalized with specific ligands for highly selective binding.[58][59][60] In affinity chromatography, the mechanism relies on reversible interactions between target molecules and immobilized ligands on the agarose matrix, enabling specific capture followed by elution. For instance, Ni-NTA agarose binds histidine-tagged proteins via coordination of the polyhistidine tag to nickel ions chelated by nitrilotriacetic acid, allowing purification from complex mixtures; elution is typically achieved using competitors like imidazole or by altering pH to disrupt the interaction.[61][62] This biospecific approach provides high purity and recovery, often exceeding 95% for recombinant proteins.[63] Agarose beads, commonly 4-6% cross-linked for enhanced mechanical stability and porosity, support linear flow rates up to 150 cm/h while maintaining resolution for proteins ranging from 10,000 to 4,000,000 Da in SEC applications.[64][65] These properties arise from the hydrophilic, inert nature of agarose, which minimizes non-specific binding and allows high sample loading capacities, typically 10-50 mg protein per mL of settled beads.[59] Specialized variants, such as those with activated groups for ligand attachment, further tailor agarose for diverse purification needs.[66] Additionally, agarose is used in immunodiffusion assays, such as the Ouchterlony double diffusion technique, where antigens and antibodies diffuse through the gel matrix to form precipitin lines at equivalence zones, enabling qualitative detection of antibody-antigen interactions. These assays, typically performed in 1-1.5% agarose gels, are applied in diagnostics for identifying specific antibodies, for example in veterinary testing for Johne's disease or avian influenza.[67][68] The introduction of Sepharose by Pharmacia in 1966 marked a pivotal advancement, replacing less stable matrices like cellulose and enabling scalable, high-resolution protein purification that transformed bioprocessing.[69]Microbiological and Cell Culture Media
Agarose serves as an effective solidifying agent in microbiological media, typically at concentrations of 1-2% w/v when mixed with nutrient broth, to create plates for bacterial cultivation and isolation. This formulation provides an inert, supportive matrix that allows bacteria such as Escherichia coli to form colonies without inhibiting growth or introducing nutritional biases, as demonstrated in high-throughput screens where agarose substrates yielded comparable microbial yields to traditional agar. Unlike agar, which contains impurities like agaropectin, purified agarose ensures consistent gelation and minimal interference with bacterial metabolism.[70] In motility studies, soft agarose gels at 0.3-0.7% w/v are employed to form gradients that facilitate the observation of bacterial chemotaxis and swarming behaviors. These low-concentration gels enable E. coli and other motile species to migrate through the matrix in response to chemical attractants, producing visible expansion rings or patterns that quantify directed movement. Such assays leverage the semi-solid nature of the gel to mimic porous environments, revealing insights into collective bacterial dynamics without the confounding effects of higher rigidity.[71][72] For cell culture applications, agarose hydrogels are widely used in tissue engineering to encapsulate mammalian cells in three-dimensional matrices, promoting viability and phenotype maintenance. Chondrocytes, for instance, encapsulated in 2-4% agarose hydrogels exhibit sustained extracellular matrix production and form cartilage-like tissues, as shown in comparative studies of hydrogel versus self-assembly methods for osteochondral grafts. This encapsulation supports nutrient diffusion and mechanical stability, enabling long-term culture of cells like human hyaline chondrocytes in models of articular cartilage repair. Agarose is also applied in neural tissue engineering, where modified agarose scaffolds, often blended with conductive polymers like aniline oligomers, support neural stem cell differentiation and axon guidance for peripheral nerve repair.[73][74][75][76] In 3D bioprinting, low-gelling temperature agarose serves as a bioink due to its shear-thinning properties and rapid gelation at around 32°C, enabling the fabrication of complex structures like vascular networks or bone scaffolds while maintaining cell viability above 90%.[77][78] Agarose hydrogels are further utilized in drug delivery systems, where their porous structure allows controlled release of therapeutics such as doxorubicin or ibuprofen through diffusion or degradation mechanisms, with release profiles tunable by concentration and crosslinking for applications in cancer therapy and inflammation treatment.[79][80] Key advantages of agarose in these contexts include its optical clarity, which facilitates high-resolution microscopy of growing colonies or encapsulated cells, outperforming agar by reducing autofluorescence and light scattering. Its biocompatibility ensures no adverse effects on microbial proliferation or mammalian cell survival, with gelling temperatures near physiological conditions (around 37°C) preserving cell integrity during encapsulation. Additionally, agarose stiffness can be tuned via concentration to achieve Young's moduli of 10-100 kPa, matching soft tissue mechanics and influencing cell behavior such as spreading and differentiation, as referenced in prior discussions of rheological properties.[70][81][82]History and Production
Discovery and Development
Agar, the precursor to agarose, has been utilized in Asia since the mid-17th century, initially as a food thickener derived from red seaweeds like Gelidium species, with its accidental discovery attributed to a Japanese innkeeper who noted the solidification of seaweed broth upon cooling in 1658.[83] Agarose itself, the neutral gelling component of agar, was first isolated in 1937 by Japanese chemist Choji Araki through acetylation of agar extracted from Gelidium amansii, marking the initial separation of this linear polysaccharide from the more charged agaropectin fraction.[2] In the 1950s, Araki advanced the understanding of agarose's structure, elucidating its composition as a repeating disaccharide unit known as agarobiose—comprising β-D-galactopyranose and 3,6-anhydro-α-L-galactopyranose—via enzymatic hydrolysis and chemical analysis of Gelidium-derived samples.[84] This structural work laid the foundation for agarose's recognition as a distinct, inert polymer suitable for biochemical applications. Following World War II, the biochemistry field's expansion, driven by discoveries like the DNA double helix in 1953, created demand for stable, transparent gels to separate macromolecules without interference, prompting Western researchers to adapt agarose for electrophoresis in the 1960s.[85] In 1961, Sten Hjertén first employed agarose as a support for zone electrophoresis, enhancing resolution over agar due to reduced electroendosmosis.[86] Early research faced challenges from impurities in natural agar extracts, such as sulfated polysaccharides and proteins from Gelidium, which clouded gels and compromised clarity during electrophoresis.[87] These issues spurred purification techniques, including alkali treatment to remove sulfate groups and improve gelling purity, as developed in the 1930s and refined post-war, enabling clearer, more reproducible agarose preparations for molecular sieving.[2]Commercial Manufacturing
Commercial production of agarose occurs on a global scale, with an estimated annual output of approximately 13,800 metric tons as of 2024, primarily derived from red algae species such as Gelidium and Gracilaria.[88] The market is dominated by a few key suppliers, including Lonza, which specializes in high-purity agarose for molecular biology applications, and Bio-Rad, a major provider of electrophoresis-grade products.[89][90] Other prominent players include Thermo Fisher Scientific and Merck (Sigma-Aldrich), which together control a significant share of the supply chain through integrated manufacturing and distribution networks.[91] The industrial manufacturing process begins with the harvesting of red algae, followed by automated alkaline extraction to isolate agar from the cell walls, where the biomass is treated with alkali solutions to depolymerize and solubilize the polysaccharides.[2] This extract undergoes filtration to remove impurities, precipitation or gelation to separate agarose fractions, and final dehydration via methods such as freeze-drying or roller-drying, though spray-drying is increasingly employed for efficient powder production at scale.[92] Quality control is rigorous, focusing on parameters like electroendosmosis (EEO), with high-grade agarose typically exhibiting low EEO values of 0.05–0.13 to ensure minimal ionic interference during electrophoresis, alongside checks for gel strength, sulfate content, and purity via spectroscopic and chromatographic analyses.[93] The agarose market is segmented primarily by application, with approximately 70% allocated to laboratory research for gel electrophoresis and nucleic acid separation, while the remaining share supports growing biotech sectors such as protein purification and biopharmaceutical development.[94] Pricing for standard molecular biology-grade agarose ranges from $800 to $1,500 per kilogram, varying by purity and volume, with bulk industrial purchases often securing lower rates around $500 per kilogram.[95][96] Sustainability efforts in agarose production emphasize shifting from wild-harvested Gelidium to cultivated Gracilaria species, which now account for over 60% of agar feedstock through aquaculture systems that reduce pressure on natural stocks and enhance supply reliability.[97] Initiatives include pond- and raft-based farming in regions like Indonesia and India, where Gracilaria yields high-quality agarose while supporting local economies and minimizing environmental impacts.[98][99] Additionally, laboratory recycling programs promote the reuse of agarose gels up to ten times, reducing waste and resource consumption in research settings.[100]References
- https://www.thermofisher.com/us/en/home/life-science/[cloning](/page/Cloning)/cloning-learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-electrophoresis-workflow.html