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. 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!

5. Spacer arm
Improve efficiency of binding
length

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 column
Target
Enzyme
Substrate analogue, cofactor, inhibitor
Immunoaffinity
Antigen, 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 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 ,2(4):

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: http://youtu.be/Hb791WsC78s

15. Thank You