Download - Affinity Chromatography
Affinity ChromatographyA Bioanalytical Tool
Guided by:
Mr. S.A.Pishwikar Sir.
Asst. Professor.
Presentation By:
Mr. Arpit H. Patel.
M.Pharm 1st sem.
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Department of Quality Assurance,Department of Quality Assurance,Bharati Vidyapeeth College Of Pharmacy, Kolhapur.Bharati Vidyapeeth College Of Pharmacy, Kolhapur.
Outlines of the Seminar:
Abbrevations.
Introduction.
Principle.
Steps of the technique.
Applications in Bioanalysis.
Recent advances.
Conclusion.
References.2
Abreviations:
GST – Glutathione S- transferace.
HIS – Polyhistidine/hexahistidine.
EDTA – Ethylene diamine tetra acetic acid.
IgG – Immunoglobulin G.
β2 AR- β2 Adrenoreceptors.
mAChR – Muscarinic acetycholine
receptors.
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Introduction:
Gel filtration Hydrophobic interaction Ion exchange Affinity
Various Biomolecule Purification Techniques.Fig No. 1.
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Various Purification Techniques.
Affinity chromatography.
• Unique purification technique.
• Separates active biomolecules.
Defination.
Liquid chromatographic technique.
biological interaction.
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Matrix Spacer arm Ligand Molecule of interest
Design of affinity chormatography model.Fig No. 2
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Components of affinity chromatography.
Principle:
Liquid adsorption chromatography.
Reversible adsorption.
Biospecific interaction.
◦Electrostatic interactions.
◦Van der Waals' forces.
◦Hydrogen bonding.
Ligand is immobilised to an matrix.
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Steps of Affinity Chromatography:
Three steps:
a) Equilibration.
b) Sample application and wash.
c) Elution.
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a) Equilibration.
• Desired conditions.
• A matrix.
• A ligand.
• Covalant coupling.
• Retention of affinity.
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Properties of Matrix.
◦An inert support.
◦Good flow properties.
◦An open pore structure.
◦Stability.
◦Degree of crosslinking.
◦ E.g. Sepharose.
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Selection of Ligand :
◦Binds reversibly.
◦Chemically compatability towards
matrix.
◦Attachment site.
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Selection of Spacer Arm:
◦Serves as a bridge.
◦Depends on situation.
◦Maximizes target binding.
The spacer arm creates a more effective environment for binding.
Fig No. 3.
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b)Sample applications and
wash.
• Mixture is poured.
• Target molecules gets binded.
• Unbound molecules are washed out .
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c) Elution
• Recovery.
• Specifically - competitive ligand.
• Non-specifically.
o buffer composition.
o Buffer pH.
o Ionic strength.
o Polarity.
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a) Equilibration.
b) Sample application and wash.
c) Elution.
Graph No. 1.
the level of complexity
qualitative information
quantitative information
total column performance
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Time
Some typical biological interactions:Glutathione - glutathione-S-transferase or
GST fusion proteins.
Metal ions - Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surfaces.
Nucleic acid - complementary base sequence, histones, nucleic acid polymerase,nucleic acid binding protein.
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Contd.Contd.
Enzyme - substrate analogue, inhibitor,
cofactor.
Lectin - polysaccharide, glycoprotein, cell
surface receptor, cell.
Hormone, vitamin- receptor, carrier
protein.
Antibody - antigen, virus, cell.
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Applications in bioanalysis:
Affinity tags
a) GST tag.
b) HIS tag.
Antibody Immobilization.
DNA binding proteins.
Screening of Active Compounds.
Purification of receptors.
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GST- tags:
Size of 220 amino acids.
High affinity for glutathione.
An expression vector.
GST-fusion protein.
◦Binding.
◦Washing.
◦Elution.
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HIS-Tags:
N- or C-terminus of the protein.
Immobilized metal affinity
chromatography(IMAC).
Transition metal ion(Cu2+, Ni2+, Zn2+,
Co2+).
Electron donar group.
Matrix-chelating agents.
21Fig 5.
Contd.
Elution
◦pH value.
◦High salt concentration.
◦High displacement agent (e.g.,
imidazole)
◦Stronger metal-chelating agent (e.g.,
EDTA)
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Protein purification process.Fig No. 4
Antibody immobilisation:
Protein A and Protein G.
Antibody binding domains.
Fc region of polyclonal and monoclonal
IgG-type antibodies.
Co-purify host IgG.
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IgG antibody.Fig No.5
DNA binding proteins:
Ability to bind DNA.
Fusion proteins.
Specific affinity for heparin.
E.g.◦ Initiation factors, ◦ Elongation factors, ◦ Restriction endonucleases, ◦ DNA ligase, ◦ DNA and RNA polymerases.
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Screening of Active Compounds:Semen Armeniacae Amarum.
◦Treats cough and asthma.
◦β2-Adrenoceptor- the main target of
drugs for cough and asthma.
◦β2-AR affinity chromatographic
stationary phase Amygdalin is
retained.
26Chinese Science Bulletin | March 2008 | vol. 53 | no. 6 |
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The chromatogram of the total extracts of Semen Armeniacae Amarum on β2-AR chromatographic column
Graph.No. 2.
Chinese Science Bulletin | March 2008 | vol. 53 | no. 6 |
Purification of receptors:
mAChRs were solubilized with digitonin.
Ligand – Dexetimide. Matrix - Carboxy N-
hydroxysuccinimide esters linked agarose beads.
Elution – Atropine.
Radioiodinated mAChRs (mAChRp) were
revealed by autoradiography.
28The EMBO Journal Vol.2 No.4: © IRL Press Limited, Oxford, England.
Recents advances:
Synthetic de novo designed ligands.◦The rational method. ◦The combinatorial method.◦The combined method.
Biomimetic ligands.
Immunospecific affinity.
Two-dimensional electrophoresis (2D-
PAGE) and mass spectrometry (MS).
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Conclusion:
A modest chance of large-scale purification of proteinaceous pharmaceuticals.
Fewer purification steps.Increased product recovery. Identification and analysis of drug
receptors. Useful in drug development.
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References:
• Affinity Chromatography-Principles and
Methods by Amersham Biosciences.
• Affinity Chromatography by Jena Biosciences.
• Chinese Science Bulletin-March 2008, vol. 53;
no. 6.
• The EMBO Journal 1982, Vol.2; No.4: © IRL
Press Limited, Oxford, England.• Google image search.
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