Overview
Affinity chromatographic separation technology is specially used for the purification of biological macromolecules. It is based on the special affinity between the fixed phase ligands and biomolecules to separate each other.
Affinity chromatography is widely used in the separation and purification of enzymes, antibodies, nucleic acids, hormones and other biological macromolecules, as well as cells, organelles, viruses and other substances. In particular, it is most effective for the separation of very little and unstable active substances, which can be purified hundreds to thousands of times by one-step affinity chromatography. For example, when isolating insulin receptors from liver cell extracts, insulin was used as ligand and coupled to AGAR carrier, which could be purified 8000 times by affinity chromatography. After decades of development, affinity chromatography has been widely used with the application of new media and the emergence of various ligands.
Principles of affinity chromatography
Affinity chromatography adsorption is mainly dependent on the biomolecule of its complementary complex (ligand) biological recognition ability, such as enzyme and substrate, antigen and antibody, hormone and receptor, nucleic acid complementary chain, polysaccharide and protein complex. Affinity chromatography is the application of biomolecular material and corresponding specific ligand molecules reversible binding principle, the ligand through covalent bond firmly bound to the solid phase of the carrier, affinity adsorption system. The conformation domains of biomolecules bind to the corresponding regions of ligands with high specificity and affinity. The binding mode is stereo conformation and has steric hindrance effect. Binding forces include electrostatic interaction, hydrophobic interaction, van der Waals forces and hydrogen bonding.
For example, the specific binding of an enzyme to a substrate is assumed to be a “multipoint binding” in which at least three functional groups in the substrate molecule should be bound to each corresponding functional group of the enzyme molecule, and this binding must hold a specific spatial configuration. That is, some of the functional groups in the substrate molecule must simultaneously maintain the configuration in which they react with the corresponding functional groups in the enzyme molecule. If the position of one of the groups concerned is changed, no more binding reactions can occur.
Will have a special affinity of a pair of any one party as a molecular ligands, under the condition of without harming its biological function, combined with insoluble carrier, immobilization, loaded into a chromatographic column, and then mixture containing purpose as mobile phase, the suitable for fixed base and purpose material under the conditions of forming complexes into the chromatographic column. At this time, in the mixture, only the target substance that can coordinate with the ligand to form the complex is adsorbed, and the impurity molecules that can’t coordinate directly flow out. After cleaning, the target product can be separated and purified by selecting appropriate eluent or changing the elution conditions to dissociate the separated substance from the fixed coordination group.
Affinity chromatography separation process
The basic process of affinity chromatography separation is as follows:
A pair of molecules with affinity to any party as ligands, under the condition of without harming its biological function, combined with insoluble carrier, curing, loaded into a chromatographic column, and then the mixture containing purpose as mobile phase, in favor of fixed matching and under the condition of goal object to form complexes into the chromatographic column. At this time, in the mixture, only the target substance that can combine with the ligand to form the complex is adsorbed, and the impurity molecules that cannot combine with the ligand directly flow out. Through the transformation of the solution composition of the column, the ligand and its affinity can be dissociated to obtain the purified product.
Application of affinity chromatography separation technology
Affinity chromatography separation technology has been widely used in various fields of life science because of its simple, rapid, specific and efficient characteristics, and has a wide application and development prospect in the field of biological products separation and analysis. Affinity chromatography is mainly used for the purification of biological macromolecules, suitable for tissue or fermentation liquid, separation of impurities and purification of the target object between the solubility, molecular size, charge distribution and other physical and chemical properties of small differences, and low relative content, other classical means of separation of difficult polymer material. Especially for the separation of some unstable polymer material is more advantageous.
1) Separation and purification of various biological molecules
Affinity chromatography can be used to separate and purify various biomolecules such as interferon, enzyme, rDNA, antigen, antibody and villus growth hormone. For example, with affinity chromatography purification of interferon, can be one step to human fibroblast interferon purification thousands of times, the separation effect is significant.
(2) Isolation and purification of various functional cells, organelles, membranes and virus particles
Affinity chromatography offers the possibility of isolating and purifying cells with different functions.
3) Used for the analysis and detection of various biological substances
Affinity chromatography has also been widely used in the analysis and detection of biological substances. For example, sheep anti-DNP (dinitrobenzene) antibodies can be detected using affinity chromatography.
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