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Shreekanta Ram

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Affinity History

1930s first developed by A.Wilhem Tiselius-a

swedish biochemist, won the Nobel Prize in1948

Used to study enzymes and other proteins.

Relies on the affinity of various biochemicalcompounds with specific properties

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Examples

Antigen Antibody

Antibody Antigen

Substrate Enzyme

DNA Histon

Hormone Binding Protein/

Receptor

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SPECIFICITY OF AFFINITY

CHROMATOGRAPHY

Specificity is based on 3 aspects.

MATRIX: for ligand attachmentSPACER ARM: use to bind ligand to

matrix

LIGAND: molecule that bindsrevesibly to a target

molecule (site of interaction)

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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 ligandwill elute off.

The substance retained in the column can beeluted off by changing the pH or organic

solvents concentration of the eluent.

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Matrix

The matrix simply provides a structure to

increase the surface area to which themolecule can bind

The matrix must be activated for the ligand tobind to it but still to retain its own activation

towards target molecules

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Matrix

Amino, hydroxyl, carbonyl & thio groups

located with the matrix serve as ligand bindingsites.

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

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Ligand

The ligand binds only to the desired moleculewithin the solution

The ligand attaches to the matrix which is madeup of an inert substance

The ligand should only interact with the desiredmolecule & from a temporary bond

The ligand/ molecule complex will remain in thecolumn, eluting everything else off

The ligand/ molecule complex dissociateschanging the pH

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Antibody affinity

(Immuno-affinity Chromatography)

Used to purify antibody against a specific antigen

Eg: Immunoglobulins

Purification of IgG, IgG 1 fragments & subclasses have thehigh affinity of protein A & protein G for the Fc regionof polyclonal & monoclonal IgG-type antibodies

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Affinity Chromatography

Can be used;

Purify & concentrate a substance from amixture into a buffering solution

Reduce the amount of substance in a mixture

Discern what biological compounds bind to aparticular substance, such as drugs

Purify & concentrate an enzyme solution

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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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u

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