Learn What is Affinity chromatography? with its principle, theory, applications, advantages and disadvantages.
What is Affinity chromatography?
Affinity chromatography is a separation technique using to purify and isolate specific biomolecules from a complex mixture. It is based on the principle of specific interactions between a target molecule and a ligand immobilized on a stationary phase. The ligand is designed to selectively bind to the target molecule, enabling its separation from other components in the mixture. Affinity chromatography have wide applications in the biotechnology industry for the purification of proteins, enzymes, antibodies, and nucleic acids.
Principle of Affinity chromatography
The principle is based on the specific binding interactions between a target molecule and a ligand immobilized on a stationary phase. The stationary phase is typically a solid support such as a resin or membrane, onto which the ligand is covalently attached.
- The ligand is a biomolecule that has a high affinity and specificity for the target molecule, such as an antibody, receptor, or enzyme.
- The target molecule is mixed with a sample containing other components and loaded onto the column. The target molecule selectively binds to the immobilized ligand on the stationary phase, while other components pass through the column.
The success of affinity chromatography depends on the specificity and affinity of the ligand for the target molecule. The ligand must be chosen carefully to ensure that it only binds to the target molecule and not to other molecules in the sample. The immobilization of the ligand onto the stationary phase must also be done carefully to ensure that the ligand maintains its binding specificity and affinity.
Theory
Affinity chromatography involves the following steps:
- Preparation of the stationary phase: The stationary phase is prepared by immobilizing the ligand onto a solid support such as a resin or membrane. The ligand is typically a biomolecule that specifically binds to the target molecule, such as an antibody or a receptor.
- Sample preparation: The sample containing the target molecule is prepared and loaded onto the stationary phase.
- Binding of the target molecule: The target molecule binds to the ligand on the stationary phase, while other components of the sample pass through the column.
- Washing: The column is washed to remove any unbound or nonspecifically bound molecules.
- Elution: The target molecule is eluted from the column using a buffer that disrupts the specific binding between the ligand and the target molecule. The eluted target molecule is then collected and further purified if necessary.
Applications
Affinity chromatography has numerous applications in the biotechnology industry, including:
- Purification of proteins: Affinity chromatography is commonly used for the purification of recombinant proteins and enzymes. The ligand can be designed to specifically bind to the target protein, enabling its separation from other proteins in the mixture.
- Purification of antibodies: For the purification of monoclonal and polyclonal antibodies. The ligand can be designed to specifically bind to the antibody, enabling its separation from other components in the mixture.
- Purification of nucleic acids: For the purification of DNA and RNA. The ligand can be designed to specifically bind to the target nucleic acid, enabling its separation from other components in the mixture.
- Drug discovery: It is a valuable tool in drug discovery for identifying and characterizing drug targets and lead compounds. The ligand can design specifically to bind the drug target, enabling the isolation and characterization of the target molecule.
Advantages
- High specificity: Affinity chromatography provides high specificity for the target molecule, enabling its purification and isolation from a complex mixture.
- High purity: Typically produces high purity products due to the specificity of the ligand for the target molecule.
- Gentle purification: It can be a gentle purification method that does not denature or damage the target molecule.
- Versatility: Can be used for a variety of biomolecules, including proteins, enzymes, antibodies, and nucleic acids.
Disadvantages
- Expensive
- Limited scale-up
- Limited specificity
- Ligand instability
In conclusion, affinity chromatography is a powerful separation technique for the purification and isolation of specific biomolecules. Its principle is based on the specific interactions between a ligand immobilized on a stationary phase and a target molecule. It has numerous applications in the biotechnology industry, including the purification of proteins, antibodies, and nucleic acids, as well as drug discovery.
Also read Principle of Paper chromatography. Spectral shifts in UV-Visible Spectroscopy.