Affinity Chromatography - Structural Biology Labs

Report
Affinity Chromatography
Supervisor:
Anton Zavialov
By:
Elham Barazeghi
Mehrafarin Ramezani
 Affinity Chromatography:
 Separation of proteins by reversible interaction
of proteins and specific ligand in matrix of
column
 Advantages:
 Selectivity
 Resolution
 Suitable for concentrating a protein
 Some common terms :
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Matrix
Spacer Arm
Ligand
Binding
Elution
Wash
Ligand coupling
Matrix
 Open and porous structure
 Inert or non-specific interaction
 OH group on sugar residues , good anchor for ligand
attachment
 Able to pass liquids through itself rapidly
 Stable enough in various conditions
 Sepharose is an ideal matrix!
Spacer arm
 Improve efficiency of binding
 length
 How it works?
 On the matrix:
 on the chromatogram:
 Essential components of Affinity
Chromatography system:
 Column type(depending on target protein and
the ligand)
 Buffers:
 Loading buffer
 Elution buffer
Elution methods:
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pH elution
Ionic strength elution
Competitive
Polarity disturbance
Denaturing
Step vs. gradient elution
Different types of Affinity
Chromatography
Type of column
Enzyme
Immunoaffinity
Lectin
Nucleic acid
Hormone, vitamin
Glutathione
Immobilized metal ion
affinity chromatography
(IMAC)
Target
Substrate analogue, cofactor, inhibitor
Antigen, virus,cell
Polysaccharides, glyco-proteins, cell surface
receptor, cell
Complementary base sequence, histones, nucleic
acid polymerase, nucleic acid binding protein
Receptor, carrier protein
Glutathione-S-transferase, GST fusion protein
Poly (his)fusion proteins, native proteins with
histidine, cystein or tryptophan residues on their
surfaces
Immuno-affinity chromatography
(IAC)
 A sub category of affinity chromatography
 A biologically related binding agent is used for the
selective purification or analysis of a target
compound
 stationary phase consists of an antibody or antibodyrelated reagent
 The selective and strong binding of antibodies for
their given targets has made them of great interest as
immobilized ligands in affinity chromatography
Moser A.C., et. Al., Immunoaffinity chromatography: an introduction to
applications and recent developments, bioanalysis -2010 ,2(4):769-790
 The loading buffer has the ability to promote fast and
efficient binding of the target analyte to immobilized
antibodies which typically occurs under physiological
conditions pH=7
 The elution carry out by temporarily lowering the effective
strength of antibody binding to the target.
 Changing the mobile phase pH is the most popular method
for eluting retained compounds from IAC columns.
 This approach is usually conducted by applying an acidic
buffer (pH 1–3) to the column
An example of competitive elution:
http://youtu.be/Hb791WsC78s
Thank You

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