AFFINITY CHROMATOGRAPHY Presented By: Salma Al-Salman

HISTORY Affinity History 1930s, first developed by A. Wilhelm Tiselius a Swedish biochemist, won the Nobel Prize in 1948 Used to study enzymes and other proteins Relies on the affinity of various biochemical compounds with specific properties

Examples Antigen Antibody Antibody Antigen Substrate Enzyme DNA Histon Hormone Binding Protein/Receptor

Specificity of Affinity Chromatography Specificity is based on three aspect of affinity 1.Matrix : for ligand attachment. 2. Spacer arm : used to bind ligand to matrix 3.Ligand : molecule that binds reversibly to a specific target molecule(site of interaction)

Mechanism of Affinity Binding Loading affinity column. Proteins sieve through matrix of affinity beads. Proteins interact with affinity ligand with some binding loosely and others tightly. Wash off proteins that do not bind.

Mechanism of Affinity Binding.. cont. Wash off proteins that bind loosely. Elute proteins that bind tightly to ligand and collect purified protein of interest.

Mechanism of Affinity Binding.. cont. The Sample is injected into the equilibrated affinity chromatography column Only the substance with affinity for the ligand are retained on the column The substance with no affinity to the ligand will elute off The substances retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent

Matrix The matrix simply provides a porous structure to increase the surface area to which the molecule can bind The matrix must be activated for the ligand to bind to it but still able to retain it’s own activation towards the target molecule

Matrix.. cont. Amino, hydroxyl, carbonyl and thiol groups located with the matrix serve as ligand binding sites Matrix are made up of agarose and other polysaccharides The matrix also must be able to withstand the decontamination process of rinsing with sodium hydroxide or urea

Ligand The Ligand binds only to the desired molecule within the solution The ligand attaches to the matrix which is made up of an inert substance The ligand should only interact with the desired molecule and form a temporary bond

ligand.. cont. The ligand/molecule complex will remain in the column, eluting everything else off The ligand/molecule complex dissociates by changing the pH

Uses Purify and concentrate a substance from a mixture into a buffering solution Reduce the amount of a substance in a mixture Discern what biological compounds bind to a particular substance, such as drugs Purify and concentrate an enzyme solution

Applications Used in Genetic Engineering Production of Vaccines And Basic Metabolic Research

ADVANTAGES OF AFFINITY CHROMATOGRAPHY Easy to achieve otherwise difficult separations Often high purity in one step Isolate pure target substance from excess amounts of contaminants Remove specific contaminants Fast separations

DISADVANTAGES OF AFFINITY CHROMATOGRAPHY 1)The interaction of proteins of interest and ligand has to be determined carefully. 2) This process required expensive materials, time, and small amount of protein that can be processed at once.

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