Affinity Chromatography - Department of Molecular and Cellular ...

Sepharose has been modified and developed to further enhance these excellent properties,
resulting in a selection of matrices chosen to suit the particular requirements for each
application (see Table 6).
In affinity chromatography the particle size and porosity are designed to maximize the
surface area available for coupling a ligand and binding the target molecule. A small mean
particle size with high porosity increases the surface area. Increasing the degree of crosslinking
of the matrix improves the chemical stability, in order to tolerate potentially harsh
elution and wash conditions, and creates a rigid matrix that can withstand high flow rates.
These high flow rates, although not always used during a separation, save considerable
time during column equilibration and cleaning procedures.
Table 6. Sepharose matrices used with Amersham Pharmacia Biotech affinity media.
Form
Mean particle size
Sepharose High Performance 6% highly cross-linked agarose 34 µm
Sepharose 6 Fast Flow 6% highly cross-linked agarose 90 µm
Sepharose 4 Fast Flow 4% highly cross-linked agarose 90 µm
Sepharose CL-6B 6% cross-linked agarose 90 µm
Sepharose CL-4B 4% cross-linked agarose 90 µm
Sepharose 6B 6% agarose 90 µm
Sepharose 4B 4% agarose 90 µm
The ligand
The ligand is the molecule that binds reversibly to a specific molecule or group of molecules,
enabling purification by affinity chromatography.
The selection of the ligand for affinity chromatography is influenced by two factors: the
ligand must exhibit specific and reversible binding affinity for the target substance(s) and it
must have chemically modifiable groups that allow it to be attached to the matrix without
destroying binding activity.
The dissociation constant (k D ) for the ligand - target complex should ideally be in the range
10 -4 to 10 -8 M in free solution.
Interactions involving dissociation constants greater than 10 -4 M, for example the binding
reaction between an enzyme and a weak inhibitor, are likely to be too weak for successful
affinity chromatography. Conversely, if the dissociation constant is lower than approximately
10 -8 M, for example the affinity between a hormone and hormone receptor, elution
of the bound substance without causing inactivation is likely to be difficult. If no information
on the strength of the binding complex is available, a trial and error approach must be used.
Refer to Appendix 7 for further details on the kinetics involved in affinity chromatography.
Altering elution methods may help to promote successful affinity chromatography when the
dissociation constant is outside the useful range (see Appendix 7).
It is important to consider the region of the ligand that will be used for attachment to the
matrix. For example, many proteins have several equivalent groups through which coupling
can take place resulting in a random orientation of the ligand on the matrix. This may
reduce the number of ligand molecules that are available in the correct orientation to bind
during an affinity purification.
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