Affinity Chromatography - Department of Molecular and Cellular ...

Avoid using magnetic stirrers as they may damage the matrix. Use mild rotation or
end-over-end stirring.
Sample preparation and application
Samples should be clear and free from particulate matter. Simple steps to clarify a sample
before beginning purification will avoid clogging the column, may reduce the need for
stringent washing procedures and can extend the life of the chromatographic medium.
Appendix 1 contains an overview of sample preparation techniques.
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.
Binding of the target protein may be made more efficient by adjusting the sample to the
composition and pH of the binding buffer: perform a buffer exchange using a desalting
column or dilute in binding buffer (see page 134).
Sample preparation techniques should ensure that components known to interfere with
binding (the interaction between the target molecule and the ligand) are removed.
Since affinity chromatography is a binding technique, the sample volume does not affect
the separation as long as conditions are chosen to ensure that the target protein binds
strongly to the ligand.
It may be necessary to test for a flow rate that gives the most efficient binding during
sample application since this parameter can vary according to the specific interaction
between the target protein and the ligand and their concentrations.
The column must be pre-equilibrated in binding buffer before beginning sample application.
For interactions with strong affinity between the ligand and the target molecule that
quickly reach equilibrium, samples can be applied at a high flow rate. However, for
interactions with weak affinity and/or slow equilibrium, a lower flow rate should be used.
The optimal flow rate to achieve efficient binding may vary according to the specific
interaction and should be determined when necessary. Further details on the kinetics
involved in binding and elution from affinity media are covered in Appendix 7.
When working with very weak affinity interactions that are slow to reach equilibrium, it may
be useful to stop the flow after applying the sample to allow more time for the interaction to
take place before continuing to wash the column. In some cases, applying the sample in
aliquots may be beneficial.
Do not begin elution of target substances until all unbound material has been washed
through the column by the binding buffer (determined by UV absorbance at 280 nm). This
will improve the purity of the eluted target substance.
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