The choice of an activated medium is dictated by both the attachable group(s) available in the ligand molecule and by the property of the binding site(s) to the given substance to be purified. It is always recommended to minimize interference with the binding site(s) of the ligand molecule. Immobilization through the least critical region of the ligand will be a good practice.

-NH₂ group to be coupled

NHS activated media is the first choice for immunoaffinity ligands due to its high coupling efficiency and long spacer arm. CNBr activated media is particularly suitable for most protein ligands. Aldehyde activated media could be used for protein ligands but the ligand affinity varies. Epoxy activated and Carboxyl activated media normally enable coupling of small ligands.

Glycosylated ligands to be coupled

Hydrazide activated media is particularly designed for the coupling of glycosylated ligands such as glycoproteins via their sugar residues that can be easily oxidized to generate formyl or ketone groups. This technique allows the immobilization to take place far away from the binding sites of the ligands. Therefore, high affinity can be maintained.

-SH group to be coupled

Irreversible Thiol-Coupling media has a long hydrophilic spacer arm. It specifically reacts with thiol group. The media can form stable and irreversible bond with thiol-containing ligands. The reaction is easy and fast. Epoxy activated media can be used to immobilize thiol-containing substances. The bond formed is very stable.

-COOH group to be coupled

Amine activated media is used to couple ligands containing carboxyl group. Stable amide bond is formed in a single reaction step.

-OH group to be coupled

Epoxy activated media is particularly useful for coupling hydroxyl-containing small molecules or large molecules.

Aldehyde or ketone group to be coupled

Hydrazide activated media is particularly useful for coupling aldehyde or ketone containing substances. Amine activated media can be used to couple aldehyde-containing molecules.

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