Affinity Chromatography
Affinity Chromatography
Dates back to 1910
Modern method was first published in 1967
Instrumental method started in 1978
Involves the interaction of a ligand with the solute of interest
Supporting
phase
Spacer arm
Ligand
Site of interaction
Affinity Chromatography
The separation is conducted in 4 basic steps
Sample
introduction
Adsorption of
components
of interest
Removal of
impurities
Retrieval of the
target compounds
= regeneration of the
column
The supporting phase
Supporting
phase
Spacer arm
Ligand
Site of interaction
The supporting phase should be rigid, stable and have a high surface area
Agarose is the most popular but cellulose and polyacrylamide can be used
Agarose can be used at moderately high pressures and over a pH range of 4-9
Cross-linking can be used to extend the pressure range
Sepharose is a bead formed of agarose gel
The spacer arm
Supporting
phase
Spacer arm
Ligand
Site of interaction
The spacer arm is a carbon chain interposed between ligand and supporting phase
Used when active site is located deep within a sample molecule
If too long, it can interact with sample species on its own (London interactions)
If too short, ligand can’t reach active site on sample molecule
The ligand
Supporting
phase
Spacer arm
Ligand
Site of interaction
Two general types of ligands
Specific
Binds only to one species
Antibody / antigen
General
Group specific
Binds to specific groups on target species
The ligand
The ligand must be covalently bound to the supporting phase to create a stable SP
Attachment can result in the ligand no longer being active
Ideal case
Binding site is
readily available
Alteration of
ligand structure
Poor orientation
or spacing
Site is not
accessible
The ligand
Samples
Types of ligands used
Enzymes
substrate, inhibitor, cofactor
Antibody
antigen, virus, cell
Lectin
polysaccharide, glycoprotein, cell receptor
Nucleic acid
polymerase,
complementary base sequence, nucleic acid,
binding protein
Hormones
receptor, carrier protein
Step elution
Since only the target protein binds,
step elution will not co-elute other sample components
Elution modes
Use of competitors
Find something that
has a stronger
affinity for your
sample than the
ligand
Elution modes
Use of competitors
Find something that
has a stronger
affinity for the ligand
than your sample
Elution modes
Use of chaotropic or denaturing agents
Change conformation
of sample
Change conformation
of ligand