Chemical Derivatives

Agarose is derivatized to adjust it's properties for various applications - especially for affinity chromotography, consult that section for aditional details and product selection.

  • Crosslinking for enhanced strength and stability:

    Agarose gels have generally been crosslinked with either epichlorhydrin or it’s analogs. As a result, proximal hydroxyl groups on adjacent strands of the double helices are covalently bridged via ether linkages and an isopropyl alcohol moiety. These crosslinks then hold the gel together under conditions that would otherwise weaken it: boiling water, freezing, high concentrations of urea, guanidine, KI or denaturing solvents like DMSO. There is NO practical reduction in the capacity of crosslinked gels to bind ligands because the “buried” hydroxyls which become crosslinked would not be available to large ligands anyway. Only those hydroxyls on the pore surfaces are available for preactivation and susequent Iigand binding.

    Note: Crosslinked agarose beads are highly recommended for shipment during the Winter months when they might freeze in transit.

  • PreActivation for coupling to ligands and Affinity Chromatography:
    • Glyoxal derivative: For binding to ligand amine groups, a “glyoxal” moiety is attached to the agarose hydroxyl groups so that it’s terminal aldehyde can readilly form a Schiff Base with a ligand amine. The Schiff Base intermediate can then be reduced to form a stable secondary amine linkage to the ligand by using sodium cyanoborohydride as shown below, where AB = agarose bead and L = Iigand:

    • Aminoethyl derivative: For binding to ligand carboxyl groups, an aminoethyl moiety is covalently attached to the agarose hydroxyl groups so that it's terminal amine can readily form an amide derivative with a Iigand carboxyl group in the presence of a suitable carbodiimide. This reaction sequence is shown below, where AB = agarose bead and L = ligand:

    • For Immobilized Metal Ion Affinity Chromatography (IMAC):

      Bidentate ligands, like imino diacetic acid (IDA) and tridentate ligands like nitrilotriacetic acid (NTA), will readily bind divalent metal ions like those shown above. Such complexes have been shown to bind histidine sequences which can be inserted as markers for expression proteins. In this way, IMAC can serve as an important technique for the identification and purification of certain expression proteins. A cost-effective source of agarose bead derivatives for IMAC can be found by clicking HERE.

    • For Ion Exchange Chromatography(IEC): For cation exchange, one can choose between carboxymethyl agarose, naturally carboxylated polysaccharide beads (like alginate), or naturally sulfated polysaccharides (like carrageenan). For anion and cation exhange, one can choose aminoethyl or DEAE derivatized agarose beads. An example of a mixed ion exchange resin would be arginine-coupled agarose beads. For more discussion of the theorry behind IEC, click on the Ion Exchange bead in the 7-Bead Application array on the home page of this website.
    • Magnetic or Hydrophobic Beads:

      See the appropriate section on this website.

References:

  1. Porath, J.(1992) Protein Express. Purif. 3, 263-281.
  2. Porath, J., Carlsson, J., Olsson, I and Belfrage, G. (1975) Nature (London) 258, 598-599.
  3. Hemdan, E. and Porath, J. (1985) J. Chromatog. 323, 255-265.

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