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 ID: 161520
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Slide1
Affinity Chromatography
Supervisor:
Anton Zavialov
By:
Elham Barazeghi
Mehrafarin RamezaniSlide2
Affinity Chromatography:
Separation of proteins by reversible interaction of proteins and specific ligand in matrix of column
Advantages:SelectivityResolutionSuitable for concentrating a proteinSlide3
Some common terms :
Matrix
Spacer ArmLigandBindingElutionWash
Ligand couplingSlide4
Open and porous structure
Inert or non-specific interaction
OH group on sugar residues , good anchor for ligand attachmentAble to pass liquids through itself rapidlyStable enough in various conditionsSepharose is an ideal matrix!
MatrixSlide5
Improve efficiency of binding
length
Spacer armSlide6
How it works?
On the matrix:
on the chromatogram:
Slide7
Essential components of Affinity Chromatography system:
Column type(depending on target protein and the ligand)
Buffers:Loading bufferElution buffer Slide8
Elution methods:
pH elution
Ionic strength elutionCompetitive Polarity disturbanceDenaturing Slide9
Step vs. gradient elutionSlide10
Different types of Affinity Chromatography
Type of column
Target EnzymeSubstrate analogue, cofactor, inhibitorImmunoaffinityAntigen,
virus,cell
Lectin
Polysaccharides, glyco-proteins, cell surface receptor, cell
Nucleic acid
Complementary base sequence, histones, nucleic
acid polymerase, nucleic acid binding protein
Hormone, vitamin
Receptor, carrier protein
Glutathione
Glutathione-S
-
transferase
, GST fusion protein
Immobilized metal ion affinity chromatography (IMAC)
Poly (his)fusion proteins, native proteins with histidine, cystein or tryptophan residues on their surfacesSlide11
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 compoundstationary phase consists of an antibody or antibody-related reagentThe selective and strong binding of antibodies for their given targets has made them of great interest
as
immobilized ligands in affinity
chromatographySlide12
Moser A.C., et. Al., Immunoaffinity chromatography: an introduction to applications and recent developments, bioanalysis -2010 ,2(4):769-790Slide13
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 columnSlide14
An example of competitive
elution: http
://youtu.be/Hb791WsC78sSlide15
Thank You