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Agarose beads consist of small spherical particles of agarose which are different in diameters and concentrations and are widely used for size exclusion or affinity chromatography. Standard, cross-linked and highly cross-linked agarose beads (without ligand linking) are used in Gel Filtration Chromatography (or size Exclusion Chromatography) in which molecules are separated by their size in solution (Figure 1).
For affinity chromatography however, ligands such as nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) are covalently linked to the agarose bead polymer which can be further used for separation of tagged proteins from other proteins (Figure 2). For decades, Agarose beads have been used for protein purification. Physical characteristics of agarose beads significantly vary between different kinds of beads and result in their different applications. Standard agarose beads (not cross-linked) are used for separation techniques whereby gravity force runs the samples through the agarose matrix. However, for high-pressure separation techniques, highly cross-linked and cross-linked beads must be chosen. Highly cross-linked and cross-linked beads are physically and chemically more stable than the standard agarose beads, hence, the higher the level of cross-linking is the more stable the matrix are. To produce Highly cross-linked and cross-linked beads, agarose beads must be treated with a particular reagent.