Affinity Chromatogra

phy

1930, First developed by A.Wilhelm Tiselius, Swedish biochemist, won the Nobel Prizein 1948

Used to study enzymes & other proteins

Relies on the affinity of various biochemical compounds with specific properities.

Affinity History

A method of separating biochemical mixtures based on a highly specific interaction between antigen & anti-body, enzyme & substrate, or receptor & hormone.

Affinity Chromatography

Antigen Antibody

Enzyme Substrate

DNA Histon

Examples:

Can be used to:

Purify & concentrate a substance from a mixture into buffering solution.

Reduce the amount of a substance in mixture

Purification of IgG fragments.

Uses:

The sample is injected into the equilibrated affinity chromatography column.

Only substance with affinity for the ligand are retained on the column.

The substance with no affinity to the ligand will elute off.

The substance retained in the column can be eluted off by changing the PH of organic solvent concentration of the eluent.

Mechanism of Affinity

Chromatography:

Used in genetic engineering , ex: nucleic acid purification.

Production of vaccines , ex: anti-body purification from blood serum.

Basic metabolic research , ex: protein or enzyme purification from cell.

Application:

Extremely high specificity.High degrees of purity can be obtained.

The process is very reproducibleThe binding sites of biological molecules can be simply investigated.

Advantages of Affinity

Chromatography:

Expensive ligands.Leakage of ligand.Degradation of the solid support.Limited lifetime.Non-specific adsorption.Relative low productivity.

Disadvantages:

Hanady KhaledHend AhmedHend BateaHend GamalHend Hassan