Avidin recovery

For the actual avidin recovery, an AML-preparation ( affinity reagent ) was used in which only one out of ten oligomer molecules carried the affinity mediator. This takes into... 

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.

Shimkus, M., Levy, J., and Herman, T. (1985) A chemically cleavable biotinylated nucleotide Usefulness in the recovery of protein-DNA complexes from avidin affinity columns. Proc. Natl. Acad. Sci. USA 82, 2593-2597. 

Fig. 11 Removal of a biotin-labeled quencher by avidin results in recovery of the MPS-PPV fluorescence.

Quantitative proteomics requires global approaches to the proteome, as prefractionation of the proteome complicates quantification due to distribution of proteins over various fractions and difficulties in determining the recovery of proteins in complex analytical procedures. However, after labelling and tryptic digestion, the tryptic peptides relevant to the study are preferentially selectively isolated from the very complex digest of the proteome. In the ICAT procedure, this is performed by avidin AfC (Ch. 18.4.1). Alternative strategies are applied, in the liquid phase based on signature peptides, or in FT-ICR-MS. 

The application of biotinylated receptor substrates is another approach, incubating the labeled substrate with the receptors prior to isolation on an avidin-coated support. In such cases, biotinylation with a cleavable biotinylation reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin would be essential for recovery of the isolated receptor. Alternatively, the receptor could be recovered by substrate competition. Perhaps one of the major drawbacks to the application of affinity techniques is the relative low molecular weight or small size of the receptor substrates, making them difficult ligands to immobilize. However, affinity procedures have been applied to the purification of a number of different receptors although relatively little work has been reported on those involved in the processing of neurotransmittors, neuropeptides, and hormones [1,2]. 

In the aforementioned work, ligands are attached covalently to a polymer. An interesting non-covalent attachment was reported early on by Wilson and Whitesides [15]. An achiral phosphine ligand linked to a biotin unit (Structure 6) interacted specifically with the protein avidin in aqueous solution to impose stereoselectivity in catalytic hydrogenation. In the rhodium-catalyzed hydrogenation of a-acetamidoacrylic acid in buffered aqueous solution, ee values of up to 40% were observed at TONs of 500 in a 48 h mn. Catalyst recovery was not a motivation for this work, but during workup the catalyst was separated from the low molecular weight products by ultrafiltration. 

The application of biotinylated receptor substrates is a useful approach, incubating the biotinylated substrates with their specific receptors prior to isolation on an avidin-coated support.In such cases, biotinylation with a cleavable reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin is essential for recovery of the isolated receptor. Alternatively, the receptor can be recovered by substrate competition. 

Figure 3.5. Effect of repeated washing of the affinity complex prior to product release on product recovery. After the initial formation and precipitation of the affinity complex from the product containing feed (S1 )> thecomplex was repeatedly thermoprecipitated and redissolved in fresh bindingbuffer (S2-S8). Afterwards the bound avidin was released in dissociation buffer (S9>S10).

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