Affinity-elution Chromatography

Fig. 1.—Affinity chromatography elution pattern for anti-rhamnose antibodies in immune serum from Streptococcus mutans. The arrows indicate the point of application of the serum and of 0.5 M L-rhamnose solution. The inset is an agar-diffusion pattern of the antibodies against the antigen. (Reprinted from Carbohydrate Research, Volume 124, J. H. Pazur, M. S. Erikson, M. Tay, and P. Z. Allen, pp. 253-263, copyright 1983, with permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK.)...

Fig. 6.—Affinity-chromatography elution patterns for the a-D- and /J-D-anti-glucose antibodies A = a-D-glucose-Sepharose 4B column, B = /3-D-glucose-Sepharose 4B column, a-G = methyl a-D-glucopyranoside, and /3-G = methyl jS-D-glucopyranoside. Insets are agar-diffusion patterns for the purified antibodies (a or /3) against a- or /3-D-glucose-BSA.

Affinity Separation - Treatment of dCTP with carbonylbis(imidazole) followed by N-trifluoroacetyl-6-aminohexyl phosphate affords (191), which was deacetylated and coupled via its aminoalkyl group to CNBr-Sepharose, affording a P4 (dC) affinity column (192).326 This column gave a one-step, 19,000-fold purification to homogeneity of deoxycytidine kinase from a crude ammonium sulphate fraction of Lactobacillus acidophilus R-26 extract, with 60 % recovery, a striking instance of the power of affinity chromatography. Elution from the column was performed with dCTP, which is a powerful end-product inhibitor for the enzyme and serves to stabilize it. 

SslA derivatives accumulated mainly in the soluble fraction of the lysed E. coli BL21 (DE3) and Rosetta Blue (DE3) cells. After isolation, the supernatant containing the S-layers was subjected to Ni affinity chromatography. Elution fractions containing the piuified proteins were pooled and dialyzed against distilled water for 12 h at 4 °C. 

Cathepsin B (from human liver) [9047-22-7] Mr 27,500 [EC 3.4.22.1]. Purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal-linked to Sepharose 4B, with elution by 2,2 -dipyridyl disulfide [Rich el al. Biochem J 235 731 1986 Methods Enzymol 80 551 1981]. 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

Affinity chromatography A form of chromatography in which separation is achieved by utilizing highly specific biochemical interactions, such as steric-or charge-related conditions, between me analyte and a molecule immobilized on a column. It is different from most forms of chromatography in mat analytes do not continuously elute from me column - only mose mat interact wim me stationary phase are retained and mus separated from omer components of me mixture under investigation. These immobilized materials are eluted from me column after all omer materials have been removed. 

Fig. 21 Chitin binding of 6xHis-tagged resilin with chitin-binding domain (6 H-resChBD) as compared to 6xHis-tagged resilin without chitin-binding domain (6 H-res). T total protein after affinity chromatography purification, B bound protein eluted from chitin beads, UB unbound protein. Reproduced from [187] with permission from The American Chemical Society, copyright 2009...

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. 

The elution of [60]- and [70]fullerenes was measured in water-methanol as a function of temperature on a poly(octadecylsiloxane) phase.67 The retention was shown to be dependent on the surface tension of the stationary phase through a simple geometrical model in which the solute formed a cavity in the stationary phase. In affinity chromatography, it was demonstrated that low ligand density may be a requirement for specificity of binding.68... 

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

Biospecific adsorption, 6 396-397, 399 Biospecific binding, in microarray fabrication, 16 385 Biospecific elution, in affinity chromatography, 6 398 Biosphere, selenium occurrence in, 22 78. 

Elovich equation, 1 595 Elution, in ion exchange, 14 410—412 Elution buffer, in affinity chromatography, 6 392... 

Nonsolvent bath, polymer precipitation by immersion in, 15 808-811 Nonspecific elution, in affinity chromatography, 6 398, 399 Nonstationary Poisson process, in reliability modeling, 26 989 Non-steady-state conduction, 9 105 Nonsteroidal antiinflamatory agents/drugs (NSAIDs) 21 231 for Alzheimer s disease, 2 820 for cancer prevention, 2 826 Nonsulfide collectors, 16 649 Nonsulfide flotation, 16 649-650 Nonsulfide mineral flotation collectors used in, 16 648-649t modifiers used in, 16 650, 651t Nonsulfide ores, 16 598, 624... 

While the individual components of the mixture may be separated from each other, they will all be contaminated to some extent with the eluting solute. Techniques such as ion-exchange chromatography and various types of adsorption and affinity chromatographies are examples of displacement methods. 

Affinity chromatography was carried out on columns prepared with lightly carboxymethylated chitin, which is known to be a poor substrate for lysozyme. Both native lysozyme and regenerated 13-105 were bound to the column at pH 7 and eluted at pH 3. As controls, the basic proteins cytochrome c and pancreatic RNase A, as well as concanavalin A and a-amylase, were not bound from the same solvent at pH 7. These findings constitute a third line of evidence for formation of native-like structure in regenerated 13-105. 

Figure 3 Biosynthesis and purification of 90-kD elastin analogue analyzed by denaturing polyacrylamide gel electrophoresis (10-15% gradient, visualized by silver staining). Lanes 1-7 time course of target protein expression at 0, 30, 60, 90, 120, 150, and 180 minutes after induction. Lane 9 soluble lysate of induced E. coli expression strain BLR(DE3)pRAMl. Lanes 10-13 protein fractions obtained from immobilized metal affinity chromatography of the lysate on nickel-NTA agarose (imidazole gradient elution). Lanes 8,14 protein molecular weight standards of 50, 75, 100, and 150 kD.

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