Introductio
n
Principle
Procedure
Elution
Types
Application
Limitation
Conclusion
Chromatography:
Chromatogr aphy is a collective ter m for a set of laboratory
techniques for the separation of mixtures .
Affinity chromatography:
Discovered by Pedro Cuatrecasas and Meir
Wilcheck.
Affinity chromatography is a type of
chromatography
specifically
that makesbind.
use of a specific affinity between
a
substance to be isolated and a molecule that
it can
Based on specific affinity
between substance to be
isolated and a molecule that it
can specifically bind(a ligand).
M + M
L L
Macromolecule Ligand (attached
to
matrices)
Complex
KINETICS IN
AFFINITY
CHROMATOGRAPHY:
COMPONENTS OF
AFFINITY
Matrix: for ligand attachment.
PHASE:-
Matrix should be
chemically and
physic ally inert.
Spacer arm: used to
.
i mprove binding between ligand
and target
b molecule o coming any effects
of
y ve Ligand: moleculeste
that binds reversibly
ricto a specific
hindrance.
r target molecule
The matrix is an inert support to which a ligand
can be directly or indirectly coupled.
It has some peculiar qualities
like:- not itself adsorb molecules to a
a. Does
significant amount.
b. Ligand must be coupled without altering its
binding properties.
c. Stability under a wide range of experimental
conditions such as high and low pH, detergent
and dissociating conditions.
The most useful matrix materials are agarose
and polyacrylamide .
S U P P O R T M AT E R I A L S TRADE NAME
Agarose Sepharose 2B,4B,6B
Cross-linked dextran Sephadex
Polyacrylamide Bio-gel-P
Cross linked cellulose Matrex Cellufine
Agarose/Polyacrylamide Ultrogel
Silica Hypersil WP 300,
Ultrasphere, Zorbax
Methacrylate Eupergit
Polystyrene Poros-50
Dextran/Polyacrylamide Sephacryl
• To prevent the attachment
of the ligand to the matrix
interfering with its ability
to bind the
• macromolecules. The
optimum length~
6-10 carbon atoms or
• their equivalent.
More commonly used for
• small immobilized ligands.
Example:-1,6-diamino
hexane and 6-amino
hexanoic acid.
COUPLIN G N A M E O F M AT R I X LIGAN D
AGENT
Cyanogen-bromide Agarose enzymes, coenzymes,
antigens, antibodies, nucleic
acid , proteins etc.
6-aminohexanoic acid;1,6- Agarose Ligands having a free amino
diaminohexane or carboxyl group
1,4-bis-(2,3- Agarose Sugars , carbohydrates,
epoxypropoxy)butane ligands with hydroxyl ,amino
or thiol group.
2- Agarose Thiol compound or sulfur
thiopyridyl containing proteins
Carboyldiimidazole Agarose N-nucleophiles
Aminoethyl ; hydrazide Polyacrylamide Carboxyl and amino ligands
The ligand is the molecule that binds
reversibly to a specific molecule or group of
molecules ,
enabling purification by
affinity
The chromatography.
selection of the ligand for affinity
chromatography is influenced by two
factors:
1. ligand must exhibit specific and
reversible bindingaffinity for the target
substance( s)
2. It must have chemically modifiable groups
that allow it to be attached
to the matrix without
destroying binding activity.
LIGAND AFFINITY
Concanavalin A Glycoproteins and polysaccharides
Calmodulin Calmodulin binding enzymes
Avidin Biotin containing enzyme
Heparin Lipoproteins, lipases , coagulation factors, DNA
polymerases , steroid receptor proteins, growth factors,
serine protease inhibiter
Proteins A and G Immunoglobins
Poly (A) RNA containing poly (U) sequences , some RNA specific
proteins
Lysine r RNA
Cibacron Blue F3G-A Nucleotide-requiring enzymes , coagulation factors
Fatty acids Fatty-acid binding proteins
Nucleotides:5’-AMP NAD+ -dependent dehydrogenases , some kinases
2’,5’ -ADP NADP+ -dependent dehydrogenases
Acriflavin Nucleotides
Addition of free ligands Introducing another protein
Lectin Affinity Chromatography
Immuno Affinity
chromatography
Metal Chelate Chromatography
Dye Ligand Chromatography
Covalent chromatography
Used for purification of glycoproteins
particularly membrane receptor proteins.
Lectins are a group of proteins produced
by plants & animals,which have
the ability to bind carbohydrates
and glycoproteins.
Used to separate mixtures of cells by taking
advantage of the saccharide components of
their outer membrane.
Commonly used lectins are:ConcanavalinA,
Soyabean lectin,etc.
Exploited in the isolation &
purification of a range of
proteins including
antigens, membrane proteins of
viral origin.
Usedfor purification of antibodies.
Ligands used is Protein A
and protirn G.
Special form of chromatography in which
an immobilised metal ions such as
Cu 2+,
Zn2+, Mn2+, Ni2+ etc. are used.
Used for purification of proteins con
a
n
tig
imidazole groups or indole groups.
Commonly metal ions are immobilised
by attachment to an
imino-diacetate or
tris(carboxymethyl)ethylenediamine
substituted agarose.
Uses a number of triazine dyes as
ligands.
Most widely used dye is Cibracron
Blue F3G-A.
Used for purification of p
iloproten
is,
interferons, coagulation factors
etc.
Developed specifically to separate thiol
containing proteins.
Most commonly used ligand is a
disulphide 2’-pyridyl group.
Used for purification of a number of
proteins but its use is limited by its cost
and rather difficult regeneration stage.
Purification of substances from biological
mixture.
Separation of native from the denatured form
of protein.
Purify and concentrate an enzyme in a
solution.
Purification of immunoglobulin.
Purification of small amount of
biological materials from high levels of
contaminating substances
Single step purification.
The matrix can be reused rapidly.
The matrix is a solid, can be easily washed
and dried.
Give purified product with high yield.
Affinity chromatography can also be used
to remove specific contaminants, such as
proteases.
Non-specific adsorption can not be
totally eliminated, it can only be
minimized.
Limited availability and high cost of
immobilized ligands.
Proteins get denatured if required pH is
not adjusted.
Affinity chromatography is one of the
most reliable
chromatographic techniques.
This technique offers high seelcv
tity
i, hence
high resolution & usually
high capacity for proteins of
interest.
It is theoretically capable of giving
absolute purifications even from
complex mixtures in a single
process.
Hand book of Affinity chromatography,
principles and Method from GE Healthcare
Practical Biochemistry, Principles and
techniques by Keith Wilson and John Walker,
Cambridge University Press
Affinity Chromatography: A Review ,Journal
of Pharmacy Research;May2011, Vol. 4
Issue 5, p1567
