Affinity chromatography, applications

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

Figure 2. An example of a typical scheme of an affinity chromatography application [1 ]...

The field of organic chemistry has seen the most extensive use of polymeric materials as aids in effecting chemical transformation and product isolations. Polymers have been used in other, related areas of chemistry. Applications have been made in analytical chemistry (pH indicators and electrode modifiers), pharmaceutical and agricultural chemistry (controlled-release drugs, pesticides, herbicides, and fertilizers), and biochemistry (enzyme immobilization and affinity chromatography). Applications of polymers to solid-phase enzymo- and radioim-mune assays (Landon, 1977 Chard, 1978) will not be discussed since they are mainly analytical in scope. 

The ELP expression system was compared to the conventional oligohistidme fusion, which is traditionally applied for purification by immobilized metal affinity chromatography (IMAC). Both techniques were shown to have a similar yield of the recombinant protein. The temperature-triggered approach offers a fast and inexpensive nonchromatographic separation with the possibility for larger scale purification. Although the ELP expression system may not be applicable to all types of recombinant proteins, numerous examples have already been shown [40]. 

Crude chloroform-methanol-water (30 60 8, v/v) extracts of immunostainedTLC bands were analyzed without further purification by nanoelectrospray low-energy mass spectrometry. The authors showed that this effective PLC/MS-joined procedure offers a wide range of applications for any carbohydrate-binding agents such as bacterial toxins, plant lectins, and others. Phenyl-boronic acid (PBA) immobilized on stationary support phases can be put to similar applications. This technology, named boronate affinity chromatography (BAC), consists of a chemical reaction of 1,2- and 1,3-diols with the bonded-phase PBA to form a stable... 

Wu, H. and Bruley, D.F, Homologous Human Blood Protein Separation Using Immobilized Metal Affinity Chromatography Protein C Separation from Prothrombin with Application to the Separation of Factor IX and Prothrombin, Biotechnol. Prog., 15, 928, 1999. 

Nickel affinity chromatography was chosen as the primary purification technique because it is a fast and reliable one-step assay and purified complexes can often be used in downstream applications without the necessity of removing the polyhistidine tag. In addition, the polyhistidine tag is smaller than many other affinity tags targeted by commercially available affinity resins and, in most cases, does not seem to interfere with the structure and function of the recombinant protein. 

Hage, D.S. (1999) Affinity chromatography A review of clinical applications. Clin. Chem. 45(5), 593-615. 

Hearn, M.T.W., Smith, P.K., Mallia, A.K., and Hermanson, G.T. (1983) Preparative and analytical applications of CDI-mediated affinity chromatography. In Affinity Chromatography and Biological Recognition, pp. 191-196. Academic Press, New York. 

Compared with enzymes fewer reports are available on immobilization of antibody (Ab) in sol-gels and their applications in immunosensing. Immobilization of Abs on a solid support was first reported in 1967 [128] and the technology has widespread application in affinity chromatography and other areas. However, the major problem associated with covalent immobilization of antibody on solid surface is partial loss of biological activity due to the random orientation of the asymmetric macromolecules,... 

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