Biotinylated molecules

Nitration of the tyrosine rings in the four binding pockets of avidin or streptavidin can be done to increase the steric hinderance within the biotin binding sites (Morag et al., 1996). This process yields chromogenic proteins that have reduced binding affinity for biotin, thus allowing elution of biotinylated molecules under mild conditions. 

The only disadvantage to the use of NHS-biotin or sulfo-NHS-biotin is the lack of a long spacer group off the valeric acid side chain. Since the binding site for biotin on avidin and streptavidin is somewhat below the surface of the proteins, some biotinylated molecules may not interact as efficiently with (strept)avidin as when longer cross-bridges are used (Green et al., 1971 Bonnard et al., 1984). 

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

Chromogenic biotinylated molecule via amide bond linkage... 

The following sections discuss the concept and use of the (strept)avidin-biotin interaction in bioconjugate techniques. Preparation of biotinylated molecules and (strept)avidin conjugates also are reviewed with suggested protocols. For a discussion of the major biotinylation reagents, see Chapter 11 and Chapter 18, Section 3. 

Since a biotinylated molecule potentially is able to interact with (strept)avidin at its biotin binding sites just as strongly as biotin in solution, the degree of biotinylation may be determined using the HABA method as well. Comparison of the response of a biotinylated protein, for example, with a standard curve of various biotin concentrations allows calculation of the molar ratio of biotin incorporation. 

Figure 8.11. A rigid to-biotinylated molecule as employed by Morgan et at. [462] to bring about formation of a self-assembled multilayer.

Suicide substrates and other biotinylated molecules must be synthesized individually... 

Avidin-gold or streptavidin-gold conjugates can be used to detect, localize, or quantify the binding of biotinylated molecules in cells, tissue sections, or blots (Morris and Saelinger, 1984 Bonnard et al., 1984 Gillitzer etal., 1990 Bronckers et al., 1987)... 

The strong binding between biotin and strept(avidin) is a disadvantage when a captured biotin or biotinylated molecule needs to be released. Release usually requires harsh conditions such as 6 M guanidine hydrochloride, pH 1.5... 

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