Affinity Chromatography - Department of Molecular and Cellular ...

Elution
There is no generally applicable elution scheme for all affinity media. Reference to
manufacturer's instructions, the scientific literature and a few simple rules should result in
an effective elution method that elutes the target protein in a concentrated form. Elution
methods may be either selective or non-selective, as shown in Figure 5.
Method 1
The simplest case. A change of buffer composition
elutes the bound substance without harming either it
or the ligand.
Method 2
Extremes of pH or high concentrations of chaotropic
agents are required for elution, but these may cause
permanent or temporary damage.
Methods 3 and 4
Specific elution by addition of a substance that
competes for binding. These methods can enhance
the specificity of media that use group-specific
ligands.
Fig. 5. Elution methods.
When substances are very tightly bound to the affinity medium, it may be useful to stop the
flow for some time after applying eluent (10 min. to 2 h is commonly used) before continuing
elution. This gives more time for dissociation to take place and thus helps to improve
recoveries of bound substances.
Selective elution methods are applied in combination with group-specific ligands whereas
non-selective elution methods are used in combination with highly specific ligands. Forces
that maintain the complex include electrostatic interactions, hydrophobic effects and
hydrogen bonding. Agents that weaken these interactions may be expected to function as
efficient eluting agents.
The optimal flow rate to achieve efficient elution may vary according to the specific
interaction and should be determined when necessary. Further details on the kinetics
involved in binding and elution of target molecules from affinity media are covered in
Appendix 7.
A compromise may have to be made between the harshness of the eluent required for elution
and the risk of denaturing the eluted material or damaging the ligand on the affinity medium.
Ready to use affinity media from Amersham Pharmacia Biotech are supplied with recommendations
for the most suitable elution buffer to reverse the interaction between the ligand
and target protein of the specific interaction. Each of these recommendations will be based
on one of the following elution methods:
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