AFFINITY CHROMATOGRAPHY Presented By: Salma Al-Salman 430204042.
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Transcript of AFFINITY CHROMATOGRAPHY Presented By: Salma Al-Salman 430204042.
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AFFINITY CHROMATOGRAPHY
Presented By:Salma Al-Salman
430204042
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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
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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 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)
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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.
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Mechanism of Affinity Binding.. cont.
Wash off proteins that bind loosely.
Elute proteins that bind tightly to ligand and collect purified protein
of interest.
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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
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Applications • Used in Genetic Engineering • Production of Vaccines
• And Basic Metabolic Research
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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
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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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