TOPIC: AFFINITY
CHROMATOGRAPHY
INTRODUCTION
● Affinity chromatography is a method of separating biochemical mixtures based on a highly
specific interaction such as that between antigen and antibody, enzyme and substrate or
receptor and ligand.
● Affinity chromatography, also known as bio selective adsorption, is a protein purification
technique. It is widely used as a means of separation and purification with specific properties.
● Biological macromolecules, such as enzymes and other proteins, interact with other molecules
with high specificity through several different types of bonds and interaction. Such interactions
include hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interaction, and
more.
● The high selectivity of affinity chromatography is caused by allowing the desired molecule to
interact with the stationary phase and be bound within the column in order to be separated from
the undesired material which will not interact and elute first.
● The molecules no longer needed are first washed away with a buffer while the desired
proteins are let go in the presence of the eluting solvent (of higher salt concentration).
PRINCIPLE
The principle of affinity chromatography is as follows:
● The stationary phase is first loaded into a column with mobile phase containing a variety of
biomolecules from DNA to proteins (depending on the purification experiment).
● Then, the two phases are allowed to bind.
● A wash buffer is then poured through a column containing both bound phases.
● The wash buffer removes non-target biomolecules by disrupting their weaker interactions with
the stationary phase.
● Target biomolecules have a much higher affinity for the stationary phase, and remain bound to
the stationary phase, not being washed away by wash buffer.
● An elution buffer is then poured through the column containing the remaining target
biomolecules.
● The elution buffer disrupts interactions between the bound target biomolecules with the
stationary to a much greater extent than the wash buffer, effectively removing the target
biomolecules.
● This purified solution contains elution buffer and target biomolecules, and is called elution
INSTRUMENTATION
The important practical requirements for affinity chromatography are as follows:
1. Matrix
● The matrix is an inert support to which a ligand can be directly or indirectly coupled.
● The most useful matrix materials are agarose and polyacrylamide.
● The matrix to be effective it must have certain characters which are as follows:
a) Matrix should be chemically and physically inert.
b) It must be insoluble in solvents and buffers employed in the process
c) It must be chemically and mechanically stable.
d) It must be easily coupled to a ligand or spacer arm onto which the ligand can be attached.
e) It must exhibit good flow properties and have a relatively large surface area for attachment.
2. Ligand
● It refers to the molecule that binds reversibly to a specific target molecule.
● The ligand can be selected only after the nature of the macromolecule to be isolated is known.
● When a hormone receptor protein is to be purified by affinity chromatography, the hormone
itself is an ideal candidate for the ligand.
● For antibody isolation, an antigen or hapten may be used as ligand.
● IF an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or effector may be used
as the immobilized ligand.
3. Solvents
● The primary buffer in affinity chromatography is the one in which the matrix resides. This
buffer should not degrade the matrix in any way. The buffer should also have a reliable effect
on the sample. The ideal buffer minimizes nonspecific interactions while maximizing the
specific interaction between the sample and the ligand.
● The other major solvent to consider in affinity chromatography is the elution buffer.
● The purpose of the elution buffer is to wash away unbound proteins initially and at higher
concentration release the desired protein from the ligand.
4. Spacer arms
● It is used to improve binding between ligand and target molecule by overcoming any effects of
steric hindrance.
● Since the success of affinity chromatography resides in its ability to bind an active site to its
corresponding ligand, if the protein binding region cannot join with the immobilized ligand the
technique is effectively useless.
STEPS INVOLVING IN AFFINITY CHROMATOGRAPHY
1. Loading of affinity column 2. Proteins sieve through matrix of affinity beads
3. Proteins interact with affinity ligand 4. Wash off proteins that do not
bind. with some binding loosely and others tightly
5. Wash off proteins that bind loosely 6. Elute proteins that bind tightly to ligand and
Collect purified protein of interest
APPLICATIONS
1. Protein purifying
Affinity chromatography has a large range of protein purifying applications. Extra
cellular and other receptor proteins can also be purified by affinity chromatography. It allows
protein purification in a relatively short amount of time with a high yield.
2. Isolation of enzyme
Enzymes can be isolated by a host at different ligands fit for bio selective adsorption.
For example, adenosine monophosphate (AMP) can be immobilized and used to bind those
proteins exhibiting an affinity for AMP, ADP, or ATP.
3. Immobilized Metal Affinity Chromatography in Proteomics
It has been proved that the progress of proteomics is mostly determined by the
development of advanced and sensitive protein separation technologies. Immobilized metal
affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich
metal-associated proteins and peptides.
4. Affinity Chromatography to The Study of Drug–Melanin Binding Interactions
Affinity chromatography using chromatographic stationary phases based on physically
adsorbed or chemically bonded melanin provides a useful tool for studying the interactions of
small molecules and metal ions with melanin
5. Purification of lectins by Bio specific affinity chromatography
Bio specific adsorbents which can be used for the purification of lectins are easily
prepared by a one-step reaction between Epoxy-activated Sepharose 6 B and Lectin-specific
sugars.
6. Purification of plasma proteins for therapeutic use
Affinity chromatography is a powerful technique for the purification of many proteins
in human plasma. It is being used in the production of various licensed therapeutic plasma
products, such as: Factor VIII, Factor IX, Von Will brand Factor, Protein C, Antithrombin III,
and Factor XI.
REFERENCES
1. Wilson, K., Walker, J. (2018). Principles and Techniques of Biochemistry and Molecular
Biology (8 eds.). Cambridge University Press: New York.
2. Instrumental methods of chemical analysis by B.K Sharma.
