Affinity Chromatography - Department of Molecular and Cellular ...

Chapter 5
Designing affinity media using pre-activated matrices
Earlier chapters in this handbook have covered a wide range of ligands that have been
coupled to Sepharose to provide ready to use affinity media for specific groups of molecules.
However, it is also possible to design new media for special purposes. When a ready to use
affinity medium is not available, a medium can be designed for the purification of one or more
target molecules by coupling a specific ligand onto a pre-activated chromatographic matrix.
For example, antibodies, antigens, enzymes, receptors, small nucleic acids or peptides can
be used as affinity ligands to enable the purification of their corresponding binding partners.
There are three key steps in the design of an affinity medium:
• Choosing the matrix.
• Choosing the ligand and spacer arm.
• Choosing the coupling method.
Choosing the matrix
Sepharose provides a macroporous matrix with high chemical and physical stability and
low non-specific adsorption to facilitate a high binding capacity and sample recovery and
to ensure resistance to potentially harsh elution and wash conditions. The choice of a preactivated
Sepharose matrix depends on the functional groups available on the ligand and
whether or not a spacer arm is required. Table 8 reviews the pre-activated matrices available.
Choosing the ligand and spacer arm
The ligand must selectively and reversibly interact with the target molecule(s) and must be
compatible with the anticipated binding and elution conditions. The ligand must carry
chemically modifiable functional groups through which it can be attached to the matrix
without loss of activity (see Table 8).
If possible, test the affinity of the ligand: target molecule interaction. Too low affinity will
result in poor yields since the target protein may wash through or leak from the column
during sample application. too high affinity will result in low yields since the target molecule
may not dissociate from the ligand during elution.
Use a ligand with the highest possible purity since the final purity of the target substance
depends on the biospecific interaction.
As discussed in Chapter 4, when using small ligands (M r < 5 000) there is a risk of steric
hindrance between the ligand and the matrix that restricts the binding of target molecules.
In this case, select a pre-activated matrix with a spacer arm. For ligands with M r > 5 000
no spacer arm is necessary.
Choosing the coupling method
Ligands are coupled via reactive functional groups such as amino, carboxyl, hydroxyl, thiol
and aldehyde moieties. In the absence of information on the location of binding sites in the
ligand, a systematic trial and error approach should be used.
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