Affinity Chromatography History and Techniques

NARESH SAH
III PHARMD
13Q1420
KCP11/4/2018

Affinity History
• 1930’s first developed by A.Wilhem Tiselius-a swedish
biochemist, won the Nobel Prize in 1948
• Used to study enzymes and other proteins.
• Relies on the affinity of various biochemical compounds with
specific properties.
• many controversies among researchers. Some say it would be
more accurate if termed bioaffinity chromatography (O'Carra
et al, 1974) or hydrophobic affinity (Shaltiel, 1974)
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• Recently, a modern form of liquid chromatography referred
to as “flash chromatography”(also known as medium
pressure chromatography) was introduced.
• The breakthrough development of affinity liquid
chromatography has enabled researchers to explore fields
such as protein–protein interactions, post translational
modifications and protein degradation that were not
possible to be examined previously.
• Finally, the coupling of reversed phase affinity
chromatography with mass spectrometry has ultimately
aided in discovery of protein biomarkers.
11/4/2018

Examples
• Antigen Antibody
• Antibody Antigen
• Substrate Enzyme
• DNA Histone(charge)
• Hormone Hormone Binding
Protein (receptor)
•MEANING
An attraction or force between particles that causes
them to combine.
The attraction between an antigen and an antibody.
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• it exploits the unique property of extremely specific
biological interactions to achieve separation and
purification.
DEF:
Affinity chromatography (AC) is a technique enabling
purification of a biomolecule with respect to biological
function or individual chemical structure.
 Very selective
 Specific binding site is used to concentrate analyte() on
column.
 to purify a particular molecule from a mixed sample.11/4/2018

Fundamental principles of affinity chromatography
 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.
 most of the proteins have an inherent recognition site
that can be used to select the appropriate affinity ligand.
The binding between the protein of interest and the
chosen ligand must be both specific and reversible
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SPECIFICITY OF AFFINITY
CHROMATOGRAPHY
• Specificity is based on 3 aspects.
MATRIX: for ligand attachment
SPACER ARM: use to bind ligand to
matrix
LIGAND: molecule that binds
revesibly to a target
molecule (site of interaction)
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• The sample is injected into the equilibrated affinity
chromatography column.
• Only the substance with affinity for the ligand are
retained on the column.
• The substance with no affinity to the ligand will elute
off.
• To release and elute the bound molecules, a desorption
step is usually performed either
• 1) specifically using a competitive ligand
or
• 2) non-specifically by changing the media atmosphere
(e.g. changing the ionic strength, pH or polarity)11/4/2018

11/4/2018
Steps of Affinity chromatography
1. Equilibration
Matrix
Specific
ligand
Equilibrate the
column and the
sample to binding
conditions.
• Prepare a gel to bind the target specifically
• Equilibrate gel and sample to binding conditions
• Apply the sample and wash out contaminants
• Desorb and elute the target
• Re-equilibrate the gel to binding conditions

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2. Sample application
3. Binding and washing
Target binds
Others
wash out
Apply sample
under binding
conditions.
Wash

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4. Desorption and elution
Target
elutes
Change the
eluent to elute
the target.

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Reconstituting
buffer
+
Changing buffer conditions
Usually decrease pH, increase ionic strength or decrease polarity adding
up to 10 % dioxane or up to 50 % ethylene glycol
Denaturing buffer
Usually extremes of pH or chaotropic agents
(A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water
molecules. This has an effect on the stability of the native state of other molecules in the solution,
mainly macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect)
General elution conditions
+

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Ligand Specificity
Protein A Fc region of many IgGs
Protein G

Competing binding substance in solution
Elution of glycoproteins from Con A Sepharose by a-D-methylmannoside
Specific eluents
+
Competing ligand in solution
Elution of enzymes from Blue Sepharose by free NADH
+
11/4/2018

• The matrix simply provides a structure to increase
the surface area to which the molecule can bind
• The matrix must be activated for the ligand to bind
to it but still to retain it’s own activation towards
target molecules.
• Amino, hydroxyl, carbonyl & thio groups located
with the matrix serve as ligand binding sites.
• Matrix are made up of agarose & other
polysaccharides
• The matrix also must be able to withstand the
decontamination process of rinsing with sodium
hydroxide or urea11/4/2018

2 types of ligand.
A)monospecific:Ab
b)groupspecific
11/4/2018

DIFFERENT AFFINITY
CHROMATOGRAPHY TECHNIQUE
(based on the types of ligand used)
1.Lectin affinity chromatography.
 Lectin potein produced by plant & animal
 Have ability to bind carbs(glycoprotein)
 Polymeric(tetrameric)
 Single specific
 Purification of glycoprotein,membrane receptor protein.
2.Immuno affinity chromatography
3.Metal chelate chromatography.
 Immoblized metal ion:cu,zn,Hg,cd/transition metal.
 Binding involves the rxn with the imidazole of histidine
residue/cysteine/indole grp of tryphtophan
 Immmobilisation is due to the formation of coordinate bond tht allows
the protein attachment and retention during the elution/non binding
contaminants.
 Elution; by lowering the PH /complexing agents:EDTA
 Ni column is used to purify recomb protein having histidine or poly
histidine tag.
 Zn column is utilized for isolation of human interference.
4.Dye ligand chromatography
5.Covalent chromatography11/4/2018
Lectins should neither be confused
with glycoproteins (proteins containing
sugar chains or residues), lecithins (fatty
substances in animals and plants),
nor leptin (the regulator of appetite and
hunger, metabolism, and behavior).

Antibody affinity
(Immuno-affinity Chromatography)
• Ab-Ag interaction.
• Ab are used as immobilized ligands
– Monoclonal antibody is linked to the agarose matrix by CNBr coupling.
• Neutral buffer/moderate salt concentration.
• Elution: high salt concenteration/urea/lowering pH ,chaeotropic agents.
• Used to purify antibody against a specific antigen
Eg: Immunoglobulins
Purification of IgG, IgG 1 fragments & subclasses have the high affinity of
protein A & protein G for the Fc region of polyclonal & monoclonal IgG-
type antibodies
11/4/2018

Affinity Chromatography
Can be used;
• Purify & concentrate a substance from a
mixture into a buffering solution
• Reduce the amount of substance in a mixture
• Discern what biological compounds bind to a
particular substance, such as drugs
• Purify & concentrate an enzyme solution
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Applications
• Used in Genetic Engineering
Nucleic acid purification
• Production Of Vaccines
antibody purification from blood serum
• And basic Metabolic Research
protein or enzyme purification from cell free extracts
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11/4/2018
1.Salting out:based on solubility of protein in salt concentration.
2.dialysis:semipermeable membrane.
3.Gel filteration:based on size.
4.IEC:based on charge.
5……………………………………………………????????????

Affinity Chromatography History and Techniques

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