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From antigen to antibody: How does affinity chromatography capture specific biomolecules?
In the field of biotechnology, affinity chromatography has attracted much attention due to its ability to separate specific biomolecules with high selectivity. This technology is based on a precise macromolecular binding interaction that can efficiently capture target molecules, thereby facilitating their extraction and purification processes.
Principles of affinity chromatography
Affinity chromatography centers on the specific binding between the target analyte (usually dissolved in the mobile phase) and its binding partner or ligand (immobilized on the stationary phase). Generally, these ligands are chemically immobilized on a solid, insoluble matrix, such as a polymer like agarose or polyacrylamide, and modified with reactive functional groups to form stable covalent bonds.
During the experiment, the loading of the solid phase and the introduction of the mobile phase are crucial. Only molecules that are effectively bound to the ligand can remain on the stationary phase.
Through a series of elution buffers and washing steps, non-target biomolecules are removed, while target molecules are retained in the solid phase and can eventually be released by elution buffer.
Affinity Chromatography Setup
Affinity chromatography can be divided into two forms: column chromatography and batch chromatography. Traditional column chromatography works by packing the solid medium into a specialized column and then passing the experimental mixture through the column for binding. Batch processing involves adding the mixture to a solid phase medium, stirring, separating, and removing the liquid phase before washing and eluting.
While column chromatography and batch processing have their own advantages and disadvantages, current technology also allows the two to be combined to achieve a more efficient process.
Application scope of affinity chromatography
Affinity chromatography excels in numerous applications, including nucleic acid purification, protein purification from extracellular extracts, and purification processes from blood. For example, affinity chromatography can be used to effectively separate proteins that bind to specific fragments and quickly obtain the desired biomolecules.
There are various types of affinity chromatography media, including amino acid media, grain protein media and scanning media, each with different uses and characteristics.
Immunoaffinity chromatography
Immunoaffinity chromatography specifically used for antibody purification is an important application of affinity chromatography. If the serum is known to contain antibodies against a specific antigen, it can be efficiently purified using this technology. This method usually uses an immobilized antigen as an affinity ligand and enjoys high specificity.
The development of immunoaffinity chromatography technology has provided a good platform for subsequent research and promoted the progress of biomedicine.
Immobilized Metal Ion Affinity Chromatography
Immobilized metal affinity chromatography (IMAC) focuses on the specific covalent bonds formed between amino acids, especially histidine, and metals. This technique allows histidine-containing proteins to be retained in a column containing immobilized metal ions and to be eluted by adjusting the pH or adding competing molecules.
Purification of recombinant proteins
Affinity chromatography also plays an important role in the purification of recombinant proteins by tagging the protein with a specific ligand to further assist in the purification process. This method can be widely used in biopharmaceuticals and research.
Application of various special media
In addition to the above applications, there are many other special media used in affinity chromatography. For example, affinity chromatography utilizing oligosaccharide binding is widely used to separate sugars or glycoproteins from proteins.
The future of affinity chromatography
Affinity chromatography technology is still evolving, and its application and benefits are still expanding with the emergence of new materials and technologies. Researchers continue to explore low-affinity-guided chromatography techniques to improve the efficiency of drug development.
In the future, how will affinity chromatography be applied to more biomedical fields to solve more complex biological problems?
