Agarose

Agarose is one of the two most popular materials used to prepare gels for use in gel electrophoresis, the other being polyacrylamide. Agarose is primarily a polymer of molecular weight approximately 120 kDa of agarobiose (an anhydrogalactose-galactose disaccharide). At sufficiently high concentrations and low temperatures, the polymer forms b-helical strands, which interact by hydrogen bonding to form the agarose gel; they dissociate at higher temperatures to form agarose solutions. Such thermally controlled gelation is the unique feature of agarose, which provides both a high reproducibility and simplicity of preparing the gel. Since gelation requires noncovalent interactions between the copolymer strands, denaturing agents, such as solutions of urea, prevent gelation.

 Agarose is derived from a natural product of oceanic algae, agar. It is processed to reduce the content of acidic groups, primarily sulfate, so as to reduce the degree of electroendosmosis that occurs during electrophoresis. Further industrial processing produces a large number of commercially available agarose fractions distinguished by (1) the degree of electroendosmosis in an electric field, determined by the number of acidic groups; (2) the degree of covalent substitution of the acidic groups with chemical groupings such as hydroxyethyl or vinyl (allyl) groups, which tends to lower the melting point in proportion to the degree of substitution; (3) the addition of linear carbohydrate polymers, such as clarified locust bean gum, to increase the viscosity of the gel and thereby eliminate measurable electroendosmosis.

Most agaroses at concentrations greater than about 0.4% (w/v) form gels at room temperature.

Agaroses with unusually high gel strengths can gel at concentrations as low as about 0.05%. The pore sizes of such agarose gels make them ideal for separating DNA molecules and oligonucleotides; their mobility is determined primarily by their size, as nucleic acids of the same class have the same charge density per nucleotide. The least concentrated gels exhibit mean pore sizes of up to 500 to 1,000 nm, sufficiently large for the penetration of large particles or small viruses (see Particle Electrophoresis). Highly soluble agarose species substituted to a maximum extent by hydroxyethyl groups can be used to prepare gels with concentrations of up to 9 to 10%. Such gels have pores of similar size to those of 3 to 6% polyacrylamide gels, and with greater resolving power for native proteins. Agarose can also be used as a copolymer with polyacrylamide, which provides larger pore sizes than polyacrylamide by itself. The copolymer is produced by adding agarose to the mixture of acrylamide monomers before its polymerization. For the separation by size of DNA fragments, or of other large particles, by capillary zone electrophoresis, solutions of agarose can be used.

 The adherence of agarose gels to glass or plastic apparatus walls is very weak, and only horizontally oriented gel electrophoresis apparatus can normally be used. The adherence of agarose to vertical glass surfaces may, however, be strengthened by drying a thin film of agarose onto glass walls; this can permit electrophoresis in vertical glass slabs or tubes. Horizontal thin-layer agarose gels with high Joule heat dissipation capacity, which are therefore amenable to electrophoresis at high field strength without melting, can be formed on hydrophilic surfaces of thin plastic sheets (eg, “Gel-Bond.( ”

 For preparative purposes, bands of a macromolecular sample in agarose gels may be recovered by solubilizing the agarose at increased temperature or adding the enzyme agarase. The macromolecule of interest is subsequently separated from the low-molecular-weight agarose fragments by filtration or precipitation methods.