Affinity Chromatography - Department of Molecular and Cellular ...

Coupling through the primary amine of a ligand
HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow
NHS-activated Sepharose is designed for the covalent coupling of ligands (often antigens or
antibodies) containing primary amino groups (the most common form of attachment) and
is the first choice for the preparation of immunospecific media. The matrix is based on highly
cross-linked agarose beads with 10-atom spacer arms (6-aminohexanoic acid) attached by
epichlorohydrin and activated by N-hydroxysuccinimide (Figure 57). Non-specific adsorption
of proteins (which can reduce binding capacity of the target protein) is negligible due to the
excellent hydrophilic properties of the base matrix. The matrix is stable at high pH to allow
stringent washing procedures (subject to the pH stability of the coupled ligand).
S
epharose
O
O CH2 CH CH2
NH (CH 2)
5 CO O N
OH
O
Fig. 57. Partial structure of NHS-activated Sepharose bearing activated spacer arms.
Ligands containing amino groups couple rapidly and spontaneously by nucleophilic attack
at the ester linkage to give a very stable amide linkage (Figure 58). The amide bond is stable
up to pH 13 making NHS-activated Sepharose suitable for applications that require
conditions at high pH.
O
S
epharose
S
epharose
O CH2 CH CH2
NH (CH 2)
5 CO O N + R NH2
OH
O
O
O CH2 CH CH2
NH (CH 2) 5 C NH R + HO N
OH
O
O
Fig. 58. Coupling a ligand to NHS-activated Sepharose.
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