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 :  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:

* * * * * 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

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 Hormone, vitamin Glutathione Immobilized metal ion affinity chromatography (IMAC) 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 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

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

  The elution carry out by temporarily lowering the effective strength of antibody binding to the target.

 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 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