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.

Most purification methods, however, involve some form of

chromatography whereby molecules in solution (mobile phase) are
separated based on differences in chemical or physical interaction
with a stationary material (solid phase). Gel filtration (also called size-
exclusion chromatography or SEC) uses a porous resin material to
separate molecules based on size (i.e., physical exclusion). In ion
exchange chromatography, molecules are separated according to the
strength of their overall ionic interaction with a solid phase material
(i.e., nonspecific interactions).
By contrast, affinity chromatography (also called affinity
purification) makes use of specific binding interactions between
molecules. A particular ligand is chemically immobilized or
“coupled” to a solid support so that when a complex mixture is passed
over the column, those molecules having specific binding affinity to
the ligand become bound. After other sample components are
washed away, the bound molecule is stripped from the support,
resulting in its purification from the original sample.

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

Affinity purification generally involves the following steps:

Incubate crude sample (e.g., cell lysate or serum) with
the affinity support to allow the target molecule in the
sample to bind to the immobilized ligand.
Wash away nonbound sample components from the
support using appropriate buffers that maintain the
binding interaction between target and ligand.
Elute (dissociate and recover) the target molecule from
the immobilized ligand by altering the buffer conditions
so that the binding interaction no longer occurs.
Use of Affinity Chromatography In Molecular Biology
Purification of mRNA:-
In order to study gene expression, molecular biologist
frequently have to isolate mRNA from total RNA preparation
which contains others types of RNA(rRNA, tRNA..) Affinity
chromatography is routinely used for this purpose. Oligo dT
is imbiolized on an agarose matrix. When total RNA
preparation is allowed to percolate through this column, only
mRNA is retarded while other RNA elute out. This becomes
possible because most mRNA molecules have a poly A tail at
their 3’-ends, recognize and bind to Oligo dT. After other
RNA molecules have eluted out, mRNA molecules bound to
the matrix are eluted by changing the wash conditions.
Isolation of DNA-binding Protein,
A transcription factor is a protein that recognize and
binds to a specific DNA sequence lying in the
regulatory region of given gene, This binding may
promote the transcription of the given gene. To study
the mechanism of action of transcription factors. It is
necessary that they be purified.
Applications
AC has been used to purify a large variety of macromolecules
such as enzymes, IG’s, membrane receptors, nucleic acids and
even polysaccharides.
Whole cells have been purified using this technique. Without
involvement of much truma, and cell remain viable.
Metal chelate affinity chromatography is a logical extension
of the basic technique.
Use of magnetic gel beads is another extension of affinity
chromatography.
Immobilized enzymes is another extension of the affinity
principle.
Thank u Google…
Picture it self speaks
References
Biophysical Chemistry – A. Upadhayay et al.
en.wikipedia.org/wiki/Affinity_chromatography
www.med.unc.edu/pharm/sondeklab/.../Affinity%20
chromatography.pdf Affinity Chromatography
Handbook
ull.chemistry.uakron.edu/chemsep/12-Affinity.pdf
Any Questions?
THANK YOU