Affinity chromatography is a method of separating biochemical mixture based on a highly specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid.

Chromatography used for separation of Antibody, cellular enzyme, and m-RNA is Affinity chromatography.
Affinity chromatography is a type of chromatographic technique that separates molecules based on their specific interactions with a ligand immobilized on a solid support. In this technique, the target molecule selectively binds to the ligand, allowing for its purification and separation from other components in the sample. Here is a detailed explanation of how affinity chromatography is used for the separation of antibody, cellular enzyme, and m-RNA:
Principle:
Affinity chromatography utilizes the principle of specific affinity between a target molecule and a ligand. The ligand is immobilized onto a solid matrix, such as agarose beads or a chromatography column, creating an affinity column.
Procedure:
1. Preparation of the affinity column:
- The ligand is immobilized onto the solid matrix through covalent bonding or physical adsorption.
- The column is then equilibrated with a buffer solution to maintain optimal conditions for binding and separation.
2. Sample application:
- The sample containing the mixture of proteins, enzymes, and m-RNA is loaded onto the affinity column.
- The target molecule, such as the antibody, cellular enzyme, or m-RNA, selectively binds to the ligand on the column.
3. Washing:
- Unbound molecules are washed away using a wash buffer, which helps remove impurities and nonspecifically bound components.
- This step ensures the purification and separation of the target molecule from other contaminants.
4. Elution:
- The target molecule is eluted from the column using an elution buffer.
- The elution buffer disrupts the specific binding between the target molecule and the ligand, allowing the target molecule to be collected separately.
5. Analysis and further processing:
- The eluted fractions containing the purified target molecule are collected and analyzed for their purity and concentration.
- Depending on the downstream applications, the purified target molecule can be further processed or used directly.
Advantages of affinity chromatography:
- High selectivity: Affinity chromatography allows for the isolation of specific target molecules based on their affinity to the ligand.
- High purity: The technique provides high purification levels, resulting in highly pure target molecules.
- Mild conditions: Affinity chromatography can be performed under mild conditions, preserving the biological activity of the target molecule.
Conclusion:
Affinity chromatography is a powerful technique used for the separation and purification of specific target molecules, such as antibodies, cellular enzymes, and m-RNA. It offers high selectivity, purity, and mild conditions, making it a valuable tool in various fields, including biochemistry, biotechnology, and pharmaceutical research.