Affinityography (Affinity Chromatography)

Introduction Affinity Chromatography is a layering technique that separates organisms using a single affinity among biomo molecules. It has long been recognized that
proteins
, enzymes and other biological large molecular substances can be combined with certain corresponding molecules single-specific and reversible, can be used for the separation and purification of biological molecules. However, due to technical limitations, mainly there is no suitable method of fixed dosing, so there is no wide application in the experiment. Until the end of the 60's, the emergence of brominated cyanide-active polysaccharide gel and coupled protein technology solved the problem of mating fixation, so that affinity layering technology has been rapidly developed. Affinity layering is the most specific and effective layering technology for separating purified proteins, enzymes and other biomolytes. It can also be used in the study of the structure and function of certain biological large molecules. Base-affinitythere are many specific interactions between biomoleleles, such as the familiar
antigen
-
antibody
, enzyme-substrate or inhibitor,
hormone
-subject, and so on, all of which are able to bind in a single and reversible way, and this binding force is called affinity. The separation principle of affinity layering is simply to separate and purify the other molecule by fixing one of the two molecules with affinity to an insoluble substitut, using the specificity and reversibility of the intermodal affinity. Molecules fixed to the substitline are called consonants, and the mats and substations are co-priced, forming a fixed phase of affinity and layering, called affinity adsorbents. Affinity layering is the first choice with the biological large molecules to be separated affinity substances as liants, such as separation enzymes can choose its substrate similars or competitive inhibitors as liants, separation antibodies can choose antigens as liants and so on. And the lid co-price is combined with the appropriate insoluble substation, such as the commonly used Sepharose-4B. The prepared affinity adsorbent is balanced, and when the sample solution passes through the affinity column, the biomolecule to be separated is specific to the mating body, thus remaining on the fixed phase, while other impurities cannot be combined with the mating body, are still in the flow phase, and flow out with the sequestration, so that only the biomolecules to be separated in the column. The purified substance to be separated is obtained by removing it from the matching body with a proper semen.
Some of the previously described layering methods, such as adsorption layering, gel
filtration
tholysis, ion exchange layering, etc. are the use of various molecular physical and chemical characteristics of differences, such as molecular adsorption properties, molecular size, molecular charged properties and so on to separate. Because of the small differences between many biological large molecules, the resolution of these methods is often low. Separating and purifying a substance usually requires a combination of methods, which not only requires more operational steps and a longer time for separation, but also reduces the recovery rate of the substance to be separated, and also affects the activity of the substance to be separated. Affinity layering is the separation and purification of biological molecules using their specific biological properties- affinity. Because of the high degree of specificity of affinity, the resolution of affinity is very high, which is an ideal method for separating biological large molecules.the selection and preparation ofaffinity adsorbent is one of the key steps in affinity and layering. It includes the choice of substate and the mating body, the activity of the substation, the association of the mating body and the substation, and so on.the properties of the (1) substitline
the substation constitutes a fixed-phase skeleton, and the affinity layering substation should have the following properties:
(1) has good physical and chemical stability. The properties of the substitum did not change significantly under the conditions of mating coupled, mating and to be separated in the process of layering, and pH and ion strength when was washed away.
(2) can be stable combination with the lid. The substitique of affinity layering should have more chemically active groups, which can be combined with the compound stable co-price through certain chemical treatment, and the basic properties of the substitum and the compound should not be changed after binding.
(3) substation structure should be a uniform porous mesh structure, so that the isolated biom molecules can be uniform, stable penetration, and fully integrated with the lid. The small aperture of the substitline will increase the resistance effect of the substitline, reduce the chance of binding between the separator and the nostril, and reduce the adsorption capacity of affinity layering. Therefore, in general, more choice of a larger aperture of the substation, so that the object to be separated has sufficient space to bind with the complex.
(4) the substitut itself and the various components in the sample are not obvious non-specific adsorption, does not affect the binding of the ligand and the subject to be separated. The substitique should have better hydromassivity in order to make the biom molecules easy to approach and act on the mating body.
General cellulose as well as cross-linked glucosaccharides,
agar
sugars, polyacrylamide, porous glass beads and other gels used for gel de-coronography can be used as a substitin of affinity and layering, of which agarose gels are most widely used. Cellulose has low price and more active groups available, but it may have obvious nonse specific adsorption effect on proteins and other biom molecules, and its stability and stability are also poor. The physical and chemical stability of cross-linked glucosamine and polyacrylamide is better, but their aperture is relatively small, and the aperture stability is not good, perhaps
Porous glass beads are characterized by good mechanical strength and good chemical stability. However, it can use less active groups, proteins and other biological molecules also have a strong adsorption effect. Agarose gel can basically better meet the above four conditions, it has a non-specific adsorption low, good stability, uniform aperture is appropriate, suitable for active and other advantages, so it has been widely used, such as Pharmacia's Sepharose-4B, 6B is currently more applications of the substitut.