Affinity Chromatography
Affinity Chromatography
Affinity Chromatography
Introduction
Affinity chromatography separates proteins on the
basis of a reversible interaction between a protein
(or group of proteins) and a specific ligand coupled
to a chromatography matrix. The technique offers
high selectivity, hence high resolution, and usually
high capacity for the protein(s) of interest.
Purification can be in the order of several
thousand fold and recoveries of active material are
generally very high.
Affinity chromatography is unique in purification
technology since it is the only technique that
enables the purification of a bio molecule on the
basis of its biological function or individual
chemical structure. Purification that would
otherwise be time-consuming, difficult or even
impossible using other techniques can often be
easily achieved with affinity chromatography. The
technique can be used to separate active bio
molecules from denatured or functionally different
forms, to isolate pure substances present at low
concentration in large volumes of crude sample
and also to remove specific contaminants.
Target Molecules and Their Ligands
• Enzyme :- substrate analogue, inhibitor, cofactor.
• Antibody :- antigen, virus, cell.
• Lectin:- polysaccharide, glycoprotein, cell surface
receptor, cell.
• Nucleic acid :- complementary base sequence, histones,
nucleic acid polymerase,nucleic acid binding protein.
• Hormone, vitamin :- receptor, carrier protein.
• Glutathione:- glutathione-S-transferase or GST fusion
proteins.
• Metal ions :- Poly (His) fusion proteins, native proteins
with histidine, cysteine and/or tryptophan residues on
their surfaces.
1. Affinity medium is equilibrated in binding buffer
2. Sample is applied under conditions that favor
specific binding of the target molecule(s) to a
complementary binding substance (the ligand). Target
substances bind specifically, but reversibly, to the
ligand and unbound material washes through the
column
3. Target protein is recovered by changing conditions to
favor elution of the bound molecules. Elution is
performed specifically, using a competitive ligand, or
non-specifically, by changing the pH, ionic strength or
polarity. Target protein is collected in a purified,
concentrated form.
4. Affinity medium is re-equilibrated with binding
buffer
Overall steps of Purification
Equilibration
adsorption of sample and elution of unbound material
Wash away unbound material
Elute bound protein(s)
Re-equilibration
Affinity Chromatography vs. Other Methods
Proteins and other macromolecules of interest can be purified from
crude extracts or other complex mixtures by a variety of methods.
Selective precipitation is perhaps the simplest method for separating
one type of macromolecule from another.
Each specific affinity system requires its own set of conditions and
presents its own peculiar challenges for a given research purpose.
Other Protein Methods articles describe the factors and conditions
associated with particular purification systems (see links in side bar
near the end of this page). Nevertheless, the general principles
involved are the same for all ligand-target binding systems, and these
concepts are the focus of this overview.
How Affinity Purification Works