Chromatography support

VI. APPLICATION OF POLYMERIC BEADS AS SIZE EXCLUSION CHROMATOGRAPHY SUPPORTS... 

Pidgeon, C., Venkataram, U. V. Immobilized artificial membrane chromatography supports composed of membrane lipids. Anal. Eiochem. 1989,... 

Alpert, A. J. and Regnier, F. E., Preparation of a porous microparticulate anion-exchange chromatography support for proteins, /. Chromatogr., 185, 375,1979. 

Succinic anhydride also is a convenient extender for creating spacer arms on chromatography supports. Supports derivatized with amine-terminal spacers may be succinylated to totally block the amine functionalities and form terminal carboxylic acid linkers for coupling amine-containing affinity ligands (Cuatrecasas, 1970). 

Glutaraldehyde also can be used to create aldehydes on amine-containing polymers. The use of this reagent in derivatizing chromatography supports and other soluble polymers is well known (Hermanson et al., 1992). 

The reactions used for coupling affinity ligands to nanoparticles or microparticles basically are the same as those used for bioconjugation of molecules or for immobilization of ligands onto surfaces or chromatography supports. However, with particles, size can be a major factor in how a reaction is performed and in its resultant reaction kinetics. Since particle types can vary from the low nanometer diameter to the micron size, there are dramatic differences in how such particles behave in solution and how the density of reactive groups or functional groups affects reactions. 

Bergseid et al. (2000) also reported on the use of SHA-activated chromatography supports for the coupling of boronate-containing affinity ligands. In this case, immobilized RNase A was used to purify anti-RNase antibodies from antiserum samples. RNase A was modified with an NHS-PDBA crosslinker at a molar ratio of 100 1 (crosslinkenprotein), purified to remove excess crosslinker, and then coupled to an SHA-agarose support in 0.1 M sodium bicarbonate, pH 8.0. 

Another potential disadvantage of an immunoaffinity separation is the assumed abundance of the purified antigen in sufficient quantities to immobilize on a chromatography support. Protein antigens should be immobilized at densities of at least 2-3 mg/ml of affinity gel to produce supports of acceptable capacity for binding antibody. Often, the antigen is too expensive or scarce to obtain in the amounts needed. 

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... 

Woodbum, K.B., Delfino, J.J., and Rao, P.S.C. Retention of hydrophobic solutes on reversed-phase liquid chromatography supports Correlation with solute topology and hydrophobicity indices, Chemosphere, 24(8) 1037-1046, 1992. 

Table 4.8. Binding affinities of immunoglobulin subclasses from different species to immobilized Protein A, Protein G, Protein L, and thiophilic chromatography supports...

M.R. Buchmeiser, New synthetic ways for the preparation of high-performance liquid chromatography supports, ). Chromatogr. A, 918 (2) 233-266, May 2001. 

The three above types occur in gas-solid chromatography on microporous adsorbents (activated charcoal, molecular sieves). Knudsen diffusion may occur in gas-liquid chromatography supports. 

Ellman s reagent has been used not only for the determination of sulfhydryls in proteins and other molecules, but also as a precolumn derivatization reagent for the separation of thiol compounds by HPLC (Kuwata et al., 1982), in the study of thiol-dependent enzymes (Masamune etal., 1989 Tsukamoto and Wakil, 1988 Alvear et al., 1989), and to create sulfhydryl-reactive chromatography supports for the coupling of affinity ligands (Jayabaskaran etal., 1987). Another important use of the compound... 

Quench the reaction by the addition of 0.1 ml of glycerol per milliliter of reaction solution. Alternatively, the reaction may be stopped by immediate gel filtration on a Sephadex G-25 column. The dextran beads of the chromatography support will react with sodium periodate to quench excess reagent. To quench the reaction with cellular samples, wash the cells with buffer to remove remaining traces of periodate. 

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