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Affinity Chromatography

What is Affinity
Chromatography???

Affinity Chromatography is a method from


which we can separate biochemical mixtures.
The separation is based on highly specific biological
interaction.
Examples:- Antigen and Antibody,
Enzyme and Subtract
By the use of Affinity Chromatography we can
separate proteins on the basis of reversible
interaction between protein and specific ligand.
This technique offers high selectivity,
high resolution and
high capacity for the protein.
Affinity Chromatography is unique purification
technology

Common terms in Affinity


Chromatography
Matrix: for ligand attachment. Matrix should
be chemically and physically inert.
Spacer arm: used to improve binding
between ligand and target molecule by
overcoming any effects of steric hindrance.
Ligand: molecule that binds reversibly to a
specific target molecule or group of target
molecules.
Binding: buffer conditions are optimized to
ensure that the target molecules interact
effectively with the ligand and are retained
by the affinity medium as all other
molecules wash through the column.

Elution: buffer conditions are changed to


reverse (weaken) the interaction
between the target molecules and the
ligand so that the target molecules can
be eluted from the column
Wash: buffers that wash unbound
substances from the column without
eluting the target molecules or that reequilibrate the column back to the
starting conditions
Ligand coupling: covalent attachment of
a ligand to a suitable pre-activated
matrix to create an affinity medium
Pre-activated matrices: matrices which
have been chemically modified to
facilitate the coupling of specific types
of ligand.

Principle of Affinity
Chromatography
Inject a sample into an initially
equilibrated affinity chromatography
column
Only the substances with affinity for the
ligand will retained in the column.
Other substances with no affinity for the
ligand will eluted from the column.
The substances retained in the column
can be eluted from the column by
changing pH or salt or organic solvent.

Method of Affinity
Chromatography
Binding of the selected ligand to the matrix requires that a

covalent bond be formed between the two which is


facilitated by derivitization of the sugar residues' hydroxyl
groups.
It is important to realize that the substrate might not be
able to reach the ligand active site if it is hidden deep
within the ligand. Therefore, most ligands are attached
first to spacer arms which are then bonded to the matrix.
The ligand-matrix gel is then loaded into an elution
column.
Once the column has been prepared, the mixture
containing isolate is poured into the elution column.
Once in the column, gravity pulls the solution through the
gel, because most of the proteins do not bind to the
ligand-matrix complex.
However, when the ligand's recognized substrate passes
through the gel, it binds to the ligand-matrix complex,
halting its passage through the gel.
Some of the impurities flow through the gel due to gravity,
but most remain, unbound, in the gel column.

In order to remove these unbound impurities, a wash of


extreme pH, salt concentration, or temperature is run
through the gel.
It is important to use a strong wash so that all the impurities
are removed, but it is also just as crucial that the wash be
not so strong that it removes the bound isolates.
Once the impurities are washed-out, the only remaining part
of the protein mixture should be the desired isolates.
Finally to collect your favorite isolate, which is still bound to
the ligand-matrix in the gel, a stronger second wash is run
through the column.
This second wash relies on the reversible binding properties
of the ligand, which allows the bound protein to dissociate
from its ligand in the presence of this stronger wash.
The protein is then free to run through the gel and be
collected.

The Chromatogram
Affinity chromatography is not just limited to isolating
one protein; given a sample of similar proteins with
similar binding affinities, a chromatogram can be
generated.
A chromatogram is a plot of absorbance vs. time for
the elution of proteins in affinity chromatography.
Four things may be learned from a chromatogram:
1)The level of complexity of the sample (indicated by
the number of peaks)
2)Qualitative information about the sample composition
(by comparing peak positions with known standards)
3)Quantitative information of the relative component
concentrations (by comparing peak areas)
4)Total column performance (by comparing with known
standards).

Chromatogram

Applications of Affinity
Chromatography
Affinity chromatography can be used to:
Purify and concentrate a substance from
a mixture into a buffering solution
Reduce the amount of a substance in a
mixture
Recognize what biological compounds
bind to a particular substance
Purify and concentrate an enzyme
solution.

Case Studies.

Determination of binding constants


byaffinity chromatography
In the use ofaffinity chromatographyto characterize biospecific
interactions in terms of reaction stoichiometry and equilibrium
constant. In that regard, the biospecificity incorporated into the
design of the experiment ensures applicability of the method
regardless of the sizes of the reacting solutes.
By the adoption of different experimental strategies
(columnchromatography, simple partition equilibrium, solidphase immunoassay and biosensor technology protocols)
quantitativeaffinity chromatographycan be used to
characterize interactions governed by an extremely broad range
of bindingaffinities.
The link between ligand-binding studies and quantitativeaffinity
chromatographyis illustrated by means of partition equilibrium
studies ofglycolytic enzymeinteractions with an exercise which
emphasizes that the same theoretical expressions apply to
naturally occurring examples ofaffinity chromatographyin the
cellular environment.

Experimental studies onaffinity


chromatographyin an electric
field

A multi compartment electrolyzer, which has been used for


preparative electrophoresis [Z. Liu, Z. Huang, J.-Y. Cong, et
al., Sep. Sci. Technol. 31 (1996) 427], is applied for carrying
outaffinity chromatographyin an alternating electric field.
The effect of electric field strength on the adsorption and
desorption characteristics is experimentally examined with
human serum albumin and Blue Sepharose Fast Flow as a
model system.

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