Affinity agarose gels

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

The affinities of antibodies for their cognate peptides can be very high, so it is sometimes difficult to recover quantitatively the higher-affinity antibodies in a polyclonal serum from the peptide resin. For this reason, it is essential to use only low concentrations of peptide on the resin (typically 100-200 pg of peptide/mL of agarose gel) and to elute bound antibody m the reverse direction to which it was run into the column, so as not to drive eluting antibodies into a further excess of antigen. If both of these criteria are adhered to, yields are usually on the order of 60-80% recovery. 

Fig. 1. IC-RT-PCR of potato latent virus. Agarose gel analysis of IC-RT-PCR assays from infected potato plants, immunocaptured using a PVS° polyclonal antibody, which also had affinity to potato latent virus (10). Lane M 100-bp molecular weight standard (SuperLadder-Low ABgene) lane 1, negative control (water) lane 2, positive control (potato virus S) and lane 3, potato latent virus.

Figure 1.3 At left is a schematic of the PCR-based method of generating DNA templates for in vitro transcription. The three-reaction method yields the DNA template shown at the bottom. Transcription from this DNA results in the desired RNA product linked to the affinity purification tag. At right is an ethidium bromide stained agarose gel showing representative results from reactions la, lb, and 2. Reprinted with permission from Edwards et al. (2009).

Total RNA or (polyA+)RNA (mRNA) can be used for experiments. In the latter case, a purification step is necessary, as mRNA is isolated from total cellular RNA by affinity chromatography on oligo-dT immobilized to a solid support. The amount of the purified RNA is determined by its dual wavelength absorbance at 260 nm and 280 nm and the quality checked by agarose gel or capillary electrophoresis. 

Of the corrinoid-agarose gels mentioned above, the corrinoids immobilized through sidechains of the corrin ring were the most effective affinity adsorbents. Although the enzyme was adsorbed com-... 

Combination of electrophoresis in different formats with affinity reactions started in the early 1950s, when Tiselius cells were used for the first time to characterise antigen-antibody interactions. A real milestone report in the field was the determination of the equilibrium constants for the binding of Ca2+ and Zn2+ to serum albumin by gel electrophoresis in 1960 [1]. Finally, the term "affinity electrophoresis" was proposed [2,3]. Its modification or, better said, its particular case, named immunoelectrophoresis, has been used for many years in reports relying on agarose gel separation in conjunction with immunoprecipita-tion. Appearance of precipitated zones on a gel is indicative for the antigen-antibody reaction and can be used for the identification of an analyte and also for its quantification. 

Cibacron Blue is a blue, polyaromatic, sulfonated dye (Fig. 6). It can be attached, as an affinity li nd, to solid matrix supjmrts (e.g. dextran, agarose) by the reaction of the triazine ring with free hydroxyl groups of the supports. The conditions of this triazine coupling method have been described by Bohme Such a dye affinity sorbent is also produced commercially, e.g. under trade name Blue Sepharose CL-6B (Cibacron Blue F3G-A covalently bound to the cross-linked agarose gel Sepharose CL-6B ) by Pharmacia, Sweden. 

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