affinity chromatography

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This PPT is describes the basics of affinity chromatoghaghy

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Shreekanta Ram

Affinity History:

Affinity History 1930’s first developed by A.Wilhem 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:

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

SPECIFICITY OF AFFINITY CHROMATOGRAPHY:

SPECIFICITY OF AFFINITY CHROMATOGRAPHY Specificity is based on 3 aspects. MATRIX : for ligand attachment SPACER ARM : use to bind ligand to matrix LIGAND : molecule that binds revesibly to a target molecule (site of interaction)

So Now What….???:

So Now What….??? 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 substance retained in the column can be eluted off by changing the pH or organic solvents concentration of the eluent .

Matrix…:

Matrix… The matrix simply provides a 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 to retain it’s own activation towards target molecules

Matrix:

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

Ligand:

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 & from a temporary bond The ligand/ molecule complex will remain in the column, eluting everything else off The ligand/ molecule complex dissociates changing the pH

Antibody affinity (Immuno-affinity Chromatography):

Antibody affinity ( Immuno -affinity Chromatography) Used to purify antibody against a specific antigen Eg : Immunoglobulins Purification of IgG, IgG 1 fragments & subclasses have the high affinity of protein A & protein G for the Fc region of polyclonal & monoclonal IgG-type antibodies

Affinity Chromatography:

Affinity Chromatography Can be used; Purify & concentrate a substance from a mixture into a buffering solution Reduce the amount of substance in a mixture Discern what biological compounds bind to a particular substance, such as drugs Purify & concentrate an enzyme solution

Applications:

Applications Used in Genetic Engineering Nucleic acid purification Production Of Vaccines antibody purification from blood serum And basic Metabolic Research protein or enzyme purification from cell free extracts

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