Transcript Affinity Chromatography - Structural Biology Labs
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