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Affinity Chromatography Supervisor: Anton Zavialov By: Elham Barazeghi Mehrafarin Ramezani.

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Presentation on theme: "Affinity Chromatography Supervisor: Anton Zavialov By: Elham Barazeghi Mehrafarin Ramezani."— Presentation transcript:

1 Affinity Chromatography Supervisor: Anton Zavialov By: Elham Barazeghi Mehrafarin Ramezani

2  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

3  Some common terms :  Matrix  Spacer Arm  Ligand  Binding  Elution  Wash  Ligand coupling

4  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! Matrix

5  Improve efficiency of binding  length Spacer arm

6  How it works?  On the matrix:  on the chromatogram:

7  Essential components of Affinity Chromatography system:  Column type(depending on target protein and the ligand)  Buffers:  Loading buffer  Elution buffer

8 Elution methods: *pH elution *Ionic strength elution *Competitive *Polarity disturbance *Denaturing

9 Step vs. gradient elution

10 Different types of Affinity Chromatography Type of columnTarget EnzymeSubstrate analogue, cofactor, inhibitor ImmunoaffinityAntigen, virus,cell LectinPolysaccharides, glyco-proteins, cell surface receptor, cell Nucleic acidComplementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein Hormone, vitaminReceptor, carrier protein GlutathioneGlutathione-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 surfaces

11 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 antibody- related reagent  The selective and strong binding of antibodies for their given targets has made them of great interest as immobilized ligands in affinity chromatography

12 Moser A.C., et. Al., Immunoaffinity chromatography: an introduction to applications and recent developments, bioanalysis -2010,2(4):769-790

13  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

14 An example of competitive elution:

15 Thank You

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