Affinity Chromatography Handbook

16
Figure 4 shows the simple procedure used to perform affinity purification on prepacked
HiTrap columns.
Fig. 4.
Equilibrate column
with
binding buffer
Apply sample
Wash with
binding buffer
3 min 5-15 min 2 min
Waste Collect
Elute with
elution buffer
Collect fractions
HiTrap columns may be used with a syringe, a peristaltic pump or a liquid chromatography
system (see Selection of Purification Equipment, Appendix 2) and are supplied with a
detailed protocol to ensure optimum results.
Media selection
A ligand already coupled to a matrix is the simplest solution. Selecting prepacked columns
such as HiTrap or HiPrep will not only be more convenient, but will also save time in
method optimization as these columns are supplied with detailed instructions for optimum
performance.
If a ligand is available, but needs to be coupled to a pre-activated matrix, refer to Chapter 5.
If no suitable ligand is available, decide whether it is worth the time and effort involved to
obtain a ligand and to develop a specific affinity medium. In many cases, it may be more
convenient to use alternative purification techniques such as ion exchange or hydrophobic
interaction chromatography.
Preparation of media and buffers
Storage solutions and preservatives should be washed away thoroughly before using any
affinity medium. Re-swell affinity media supplied as freeze-dried powders in the correct
buffer as recommended by the manufacturer.
Use high quality water and chemicals. Solutions should be filtered through 0.45 µm or
0.22 µm filters.
Reuse of affinity media depends on the nature of the sample and should only be considered
when processing identical samples to avoid cross-contamination.
If an affinity medium is to be used routinely, care must be taken to ensure that any
contaminants from the crude sample can be removed by procedures that do not damage
the ligand.
Binding and elution buffers are specific for each affinity medium since it is their influence
on the interaction between the target molecule and the ligand that facilitates the affinitybased
separation. Some affinity media may also require a specific buffer in order to make
the medium ready for use again.