Agarose

Agarose is also a natural polysaccharide (galactan), extracted from seeweed [95]. The basic repeating unit (agarobiose) is given in Fig. 17. 

This formula, however, is somewhat idealized, and natural agaroses always have some degr of substitution by sulfate, pyruvate and methoxyl groups. 

The pores produced by the association of these polymer chains are sufficiently large to enable the diffusion of biological macromolecules into the matrix but because the chains are not covalently cross-linked the mechanical and chemical stabilities are not good and the particles degrade over a period of continuous use. To improve the mechanical and chemical stability of the matrix, individual polymer chains may be cross-linked by treatment of the non-cross-linked bead with epichlorohydrin, diepoxides or divinylsulphone [11]. As the cross-linking is between associated polymer chains there is no reduction in the pore size or permeability of a bead when cross-linking occurs. This is the basis of the commercial product Sepharose CL (Pharmacia). 

Agarose being hydrophilic and highly permeable is ideally suited for size exclusion chromatography of biological macromolecules. Also the availability of numerous 

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There are several forms of electrophoresis. In slab gel electrophoresis the conducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs are formed by pouring the gel between two glass plates separated by spacers. Typical thicknesses are 0.25-1 mm. Gel electrophoresis is an important technique in biochemistry, in which it is frequently used for DNA sequencing. Although it is a powerful tool for the qualitative analysis of complex mixtures, it is less useful for quantitative work. 

An example of a size-exclusion chromatogram is given in Figure 7 for both a bench-scale (23.5 mL column) separation and a large-scale (86,000 mL column) mn. The stationary phase is Sepharose CL-6B, a cross-linked agarose with a nominal molecular weight range of 5000-2 x 10 (see Fig. 6) (31). 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

Distinction is also made among electrophoretic techniques in terms of the type of matrix employed for analysis. Matrices include polymer gels such as agarose and polyacrjiamide, paper, capillaries, and flowing buffers. Each matrix is used for different types of mixtures, and each has unique advantages. 

Some forms of agarose are specifically designed to work with large (mol wt >500,000) molecules (27,28). The types of samples for which the agarose ief system are utilized are larger plasma proteins such as immunoglobulins, tissues, and tumors. 

Another form of ief is a method called direct tissue isoelectric focusing (dtif) (29) where isoelectric focusing in agarose is used to evaluate tissues. 

Various support media may be employed in electrophoretic techniques. Separation on agarose, acrylamide, and paper is influenced not only by electrophoretic mobiUty, but also by sieving of the samples through the polymer mesh. The finer the weave of selected matrix, the slower a molecule travels. Therefore, molecular weight or molecular length, as well as charge, can influence the rate of migration. 

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Both the ease of use of this method for characterization of proteins and nucleic acids, and the abiHty to analyze many samples simultaneously for comparative purposes, have led to the prevalence of this technique. The drawbacks of a polyacrylamide matrix is that acrylamide is a neurotoxin, the reagents must be combined extremely carefiiUy, and the gels are not as pHable as most agarose gels. 

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 p.m capillary, for example, there is 0.03 P-L/cm capillary length. This is 1/100 to 1/1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or duorescence (47,48). The conductivity detector claims detection limits down to lO molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace AND RESIDUE ANALYSIS). 

Amido black is a commonly used stain, but it is not very sensitive. It is often used to visualize concentrated proteins or components that are readily accessible to dyes such as proteins that have been transferred from a gel to nitrocellulose paper. Two of the more sensitive and more frequently used stains are Coomassie Brilliant Blue (R250 and G250) and silver stains. Because these stains interact differently with a variety of protein molecules, optimization of the fixative and staining solutions is necessary. The Coomassie stains are approximately five times more sensitive than amido black and are appropriate for both agarose and polyacrylamide gels. The silver stain is approximately 100 times more sensitive than Coomassie and is typically used for polyacrylamide gels. 

The double-immunodiffusion technique, often referred to as the Ouchtedony technique, uses an agarose gel as the matrix. Holes are made in the agarose where either sample or antisera is placed. The two solutions are allowed to diffuse into the matrix for a predetermined time. If there is a reaction... 

At a somewhat more basic level, both agarose and acrylamide gel systems have been used for direct immunofixation. In these gels, samples are electrophoresed and then immunofixed by either using stnps of cellulose acetate soaked in an antibody or the antibody is placed direcdy over the sample area of the gel. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Blotting techniques may be used in a variety and combination of electrophoretic systems which makes their use widespread and convenient when protein concentrations are minimal and agarose or polyacrylamide is the matrix choice. 

Plus One REPEL-SILANE ES (a solution of 2% w/v of dichloromethyl silane in octamethyl cyclooctasilane) is used to inhibit the sticking of polyacrylamide gels, agarose gels and nucleic acids to glass surfaces and is available commercially (Amersham Biosciences). 

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