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Affinity chromatography was introduced almost 50 years back as a
powerful tool to separate biologically active molecules like protein
from a mixture.
This was discovered by Pedro Cuatrecasas and Meir Wilcheck in
It uses a unique property of extremely specific biological interactions
to achieve separation and purification.
Binding to ligand:- the crude sample with affinity support is
incubated and allow the target molecule to bind to immobilized
Separation:-Washing away of non-bound sample from the support.
Elution :- the target molecule is eluted from the immobilized ligand
by altering the buffer conditions which interrupts the binding.
Affinity chromatography is an excellent method to purify or separate a target
molecule from a mixture.
Theoretically, this method is capable of giving absolute purification, even from
complex mixtures, in a single process.
It was originally developed for the purification of enzymes, but it has been
extended to nucleotides , nucleic acids, immunoglobulins, membrane receptors
and even to whole cells and cell fragments.
This method is highly based on specific interactions between the molecules.
These interactions which are typically reversible are used for purification by
placing one of the interacting molecule referred to as affinity ligand onto a solid
matrix which will the stationary phase and the target molecules in the mobile
Matrix : for ligand attachment. The matrix
should be chemically and physically inert.
Spacer arm : used to improve the binding
between the ligand and the target molecule
by over coming the affect of steric hindrance.
Ligand : the molecule the binds reversibly to
a specific target molecule or group of target
Matrix is a substance used, usually in a bead form to which a specific ligand is
1. It must be insoluble in the solvents and the buffers used.
2. Should possess suitable and sufficient chemical groups to which the ligand may
be covalently coupled, and be stable under the conditions of the attachment.
3. Be stable during binding of the macromolecule and its subsequent elution.
4. Exhibit good flow properties.
Sepharose , a bead-form of agarose provides these properties.
Ligand is the molecule that binds specifically to the target molecule or a group of
molecules, enabling purification by the process.
2 factors important for the selection of ligand:
1. The ligand must exhibit specific and reverse binding affinity for the target
2. It must have chemically modifiable groups that allow it to the matrix to be
attached to the matrix without destroying binding activity.
The dissociation constant (Kd) for the ligand –target complex should ideally be in
the range 10-4 (enzyme) and 10-8(hormone).
Altering elution method may help to promote successful affinity chromatography
when the dissociation constant which is outside the useful range.
If several functional groups are available, couple the ligand least likely to be
involved in the specific affinity reaction. A range of pre-activated matrices for
attachment of the ligand through different functional groups are available
The binding site of a target protein is located
deep within the molecule and an affinity
medium prepared by coupling small ligands
such as enzyme co-factors, directly
Sepharose may exhibit low binding capacity
due to steric interference i.e. the ligand is
unable to access the binding site of the
In these circumstances the spacer arm is
interposed between the matrix and the
ligand to facilitate effective binding.
Spacer arms must be designed to maximize
binding, but to avoid non-specific binding
The size and length of the spacer arm is very
1. Preparation of column: The solid support (bead matrix) is a gel loaded into an
Sepharose, agarose and cellulose are the most commonly used solid support ,
because the hydroxyl groups on the sugar residues can be easily manipulated to
accept a ligand.
The ligand is selected according to the desired isolate.
2. Loading the solution containing the target molecule: the solution is usually a
protein rich mixture is poured into the elution column and allowed to run through
the gel , at a controlled rate.
The target molecule specific for the ligand will covalently bind and the other
molecules flowing through.
This follows the washing of the column with buffer to remove the unbound
• Method 1: In simplest
cases, a change of buffer
composition elutes the
target molecule which is
• Method 2: extremes of pH
or high concentrations
chaotropic agents are
required for elution, but
these may cause
permanent or temporary
• Method 3 and 4: Specific
elution by the addition of
substance that competes
for binding. These
methods can enhance the
specificity of the media
that use group specific