Affinity chromatography supports

Figure 20.15 An affinity chromatography support containing iminodiacetic acid groups chelated with nickel may be used to remove excess enzyme after reactions to produce antibody-enzyme conjugates. The nickel chelate binds to the antibody in the Fc region, retaining the conjugate while allowing free enzyme to pass through the gel unretarded.

High performance adhesives, 1 545 High performance affinity chromatography support, 6 394-395... 

Narayanan, S. R., and Crane, L. J. (1990). Affinity chromatography supports A look at performance requirements. Trends Biotechnol. 8, 12-16. 

Sinninger, V. Tapon-Bretaudiere, J. Zhou, F.L. Bros, A. et al. Immobihzation of dermatan sulphate on a sihca matrix and its possible use as an affinity chromatography support for heparin cofactor II purification. J. Chromatogr. 1991, 539, 289. 

The isolation of mRNA also became possible during the years of the decade. The discovery that a large proportion of mRNA species carried polymers of adenylate at their 3 -OH ends enabled the design of affinity chromatography supports such as oligo-dT cellulose and poly-U Sepharose which, because of their complementarity with the poly-A tails of the mRNA, could be employed to purify them from total RNA. These same poly-A tails made the purified mRNAs ideal substrates for reverse transcriptase. Oligomers of deoxythymidine (dT) could hydrogen bond to the poly-A and provide a primer for the initiation of transcription of a cDNA. 

Although most of the biopolymers used as affinity chromatography supports are polysaccharides, other biomolecules also have been reported. Purification of... 

Chromatography matrices. DEAE-cellulose (DE-52, Whatman) hydroxylapatite (Bio-gel HTP, Bio-Rad), Mono Q anion exchange columns (Pharmacia Fine Chemicals) and Ni-NTA nickel affinity chromatography support (Qiagen)... 

For Yiv > YPv> where y v and Ypv are the surface tensions of liquid and protein, respectively, AFads increases with increasing ysv, predicting decreasing polymer adsorption. An example of this is phosphate buffer saline where y]v = 72.9 mJ/m2 and Ypv is usually between 65 and 70mJ/m2 for most proteins [5]. Therefore, supports for gel-permeation and affinity chromatography should be as hydrophilic as possible in order to minimize undesirable adsorption effects. 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

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... 

Affinity chromatography involves precisely the same kind of electrostatic, hydrophobic, dipolar, and hydrogen-bonding interactions described above, but the specificity of binding is extraordinarily high. Demands on the homogeneity of the stationary phase and on the rigidity of the support are often... 

Some kinds of chromatography require relatively little optimization. In gel permeation chromatography, for example, once the pore size of the support and number of columns is selected, it is only rarely necessary to examine in depth factors such as solvent composition, temperature, and flow rate. Optimization of affinity chromatography is similarly straightforward. In RPLC or IEC, however, retention is a complex and sensitive function of mobile phase composition column type, efficiency, and length flow rate gradient rate and temperature. 

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