Affinity biotinylation approach

Confocal laser scanning fluorescence microscopy was used to study the exposure of the avidin-specific binding sites in the Av-GEB platform by the immobilization of a small and flexible biotinylated fluorescein molecule as a fluorescence marker. Fluorescence microscopy thus confirms that Av-GEB platform exposes active binding sites for biotin, acting as affinity matrix (Fig. 21.2B). After use, the electrode surface can be renewed by a simple polishing procedure for further uses, highlighting a clear advantage of this new material with respect to surface-modified approaches such as classical biosensors and other common... 

In an approach similar to the cell-like compartments, Doi and Yanagawa (1999) used biotinylated DNA to display peptides fused to streptavidin in compartments of water in oil emulsions. The method was named streptavidin-biotin linkage in emulsions, STABLE (Doi and Yanagawa, 1999). Upon in vitro translation each translated peptide is displayed as a fusion to streptavidin that binds to its encoding biotinylated DNA in its compartment. The resulting protein-DNA fusions can then be recovered and used for affinity selection. To avoid cross-contamination, biotin has to be added before recovery because much more streptavidin will be produced in each compartment than biotinylated DNA is present. The selected DNA-protein complexes can then be amplified by PCR. The principle of this selection strategy is shown in Figure 7. 

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]. 

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