Affinity spacer

Macromolecules bearing reactive groups in the repeat units along their chains are capable of multiple interaction with the matrix. As early as 1973, Wilchek prepared Sepharose-based supports chemically modified by chemisorbed polylysine and polyvinylamine [41]. The leakage of dyes covalently bonded to these supports was reduced remarkably as compared to non-modified Sepharose activated by cyanogen bromide. Thus, stable and high capacity affinity adsorbents could be prepared by the introduction of macromolecular spacers between a matrix and a biospecific ligand. 

Compounds 13 and 20 were obtained in five and seven steps respectively from the intermediate alcohol 6. They, as well as the acetylated derivative 12, were tested in vivo i and proved to be, as their free acids, gratuitous inducers of pectinases in Erwinia chiysanthemi. These results provide evidence that the hydroxyl function in C-5 is not required for the recognition between inducers and the KdgR repressor protein. Tharefore we can now envisage the preparation of affinity columns, by immoWlizing inducers on suppc rts using a spacer arm boimd to the C-5 hydroxyl. Such columns could then be used for the purification of the repressor protein. 

Huot et al. [38] used affinity chromatography to identify and partially purify an amiloride-binding protein with characteristics of the renal brush border Na /H exchanger. The high-affinity amiloride analog A35 (5-A-(3-aminophenyl)amiloride) was coupled to Sepharose CL-4B through a triglycine spacer. Rabbit renal brush border membranes were solubilized with 0.6% Triton X-100, incubated with the... 

Matrix Attached protein or affinity ligand, or spacer and affinity ligand Ref. 

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). 

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled peptides from a (strept)avidin affinity column. The use of 14C isotopes instead of deuterium labels permits precise reverse phase separations prior to mass spec that show no elution peak time differences between isotope-labeled and normal atom-labeled peptides.

Figure 16.5 A catch-and-release ICAT design incorporates a gem-methyl group and an isopropyl group on either side of a disulfide bond within its spacer arm. The hindered disulfide permits the use of standard reducing gel electrophoresis conditions using DTT without reduction. After purification on a (strept)avidin affinity column, however, the disulfide group can be cleaved with TCEP, which provides recovery of the labeled peptides prior to mass spec separation.

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.

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