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Contents
• Introduction
• Principle
• Steps
• Specificity
• Different types of Affinity Chromatography
• Applications
• Advantages
• Disadvantages
 Introduction
• Purification of a specific molecule or a group
of molecules from complex mixtures through
its specific binding properties to an
immobilized ligand.
Ligand protein interaction
Ligand Protein of interest

Matrix

Impurities
Outline of Affinity Chromatography
 Principle
• Separation of a desired protein using affinity
chromatography relies on the reversible
interactions between the protein to be
purified and the affinity ligand coupled to
chromatographic matrix.
Principle of affinity chromatography

(a). Sample (Substance to be isolated + “impurities”) application

(b). Immobilized ligand specifically absorbs the substance to be isolated
(c). Elution of the “impurities”
(d) Desorbtion of the specific-bound substance
 Three main steps
• Binding of protein to ligand
• Washing of unwanted impurities or proteins
• Elusion of protein of interest
 Steps of Affinity Chromatography
• The column and stationary phase are flushed with
a buffer solution to prepare the ligands for
binding
• The sample is introduced under conditions that
favour the binding of the target molecules to the
affinity ligands. The target molecules bind to the
ligands and the unbound molecules are washed
away.
• By changing the elution conditions or using
another ligand, the target molecules are freed
from the ligands and elute from the column giving
a purified protein.
Specificity of Affinity Chromatography
• Is based on three aspects:
• Matrix
• Spacer
• Ligand
 Matrix
• The matrix simply provides a structure to
increase the surface area to which the molecule
can bind.
• Amino, hydroxyl and carbonyl groups located
with the matrix serve as ligand binding sites.
• Matrix are made up of agarose and other
polysaccharides.
 Properties of matrix
• For having an effective matrix, it must have
certain characters:
1). It must be insoluble in solvents and buffers
employed in the process
2). It must be chemically and mechanically stable.
3). It must be easily coupled to a ligand or spacer
arm onto which the ligand can be attached.
4). It must exhibit good flow properties and have a
relatively large surface area for attachment
 Spacer arm
• The stationary phase is typically a gel matrix,
to prevent steric interference or overlap
during the binding process of the target
molecule to the ligand, an inhibitor containing
a hydrocarbon chain is first attached to the
agarose bead (solid support).
• This inhibitor with a hydrocarbon chain is
commonly known as the spacer between the
agarose bead and the target molecule.
Spacer Arm
 Ligands
• The Ligand binds only to the desired molecule
within the solution.
• It attaches to the matrix which is made up of an
inert substance.
• It should only interact with the desired molecule
and form a temporary bond.
• The ligand/molecule complex will remain in the
column, eluting everything else off.
• The ligand/molecule complex dissociates by
changing the pH.
 Different types of affinity
chromatography
Type of column Target
Enzymes Substrate analogue, inhibitor, Cofactor.
Immunoaffinity Antigen
Lectin Carbohydrates, cell surface receptors.
Nucleic acid Complementary base sequence, histone
Hormone Receptor or carrier protein
Glutathione Glutathione-S-transferse
Immobilized Metal ion Affinity Poly(histidine) Fusion proteins
Chromatography
Purification of Recombinant protein
Affinity Chromatography Purification
 Applications
• Used in Genetic Engineering
Nucleic acid purification
• Production of Vaccines
Antibody purification from blood serum
• Basic Metabolic Research
Protein or enzyme purification from cell
free extracts
Advantages of affinity chromatography
• Extremely high specificity
• High degrees of purity can be obtained
• The process is very reproducible
• The binding sites of biological molecules can
be simply investigated
 Disadvantages
• Expensive ligands
• Leakage of ligand
• Degradation of the solid support
• Limited lifetime
• Non-specific adsorption