Streptavidin-biotin binding interaction

We selected the streptavidin-biotin binding interaction for proof-of-concept studies of the immobilized nanoSPR sensor for the following reasons first, because of its very high association constant, streptavidin-biotin binding is insensitive to washing steps, which simplifies the experimental setup. Second, the wide use of this model system for validation of other biosensors enables comparison of the nanoSPR biosensor with other platforms under development. ... 

Figure 18.14 NHS-SS-PEG4-biotin can be used to label a primary antibody molecule that has specificity for a protein or interest. Incubation of the biotinylated antibody with a sample, such as a cell lysate, allows the antibody to bind to its target. Capture of the antibody-antigen complex on an immobilized streptavidin reagent effectively isolates the targeted protein from the other proteins in the sample. The disulfide linkage in the spacer arm of the biotin tag permits elution of the immune complex from the streptavidin support using DTT and without using the strong denaturing condition typically required to break the streptavidin-biotin interaction.

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

Biotin s interaction with the proteins avidin and streptavidin is among the strongest noncovalent affinities known (K l = 1015 M l). The binding occurs between the bicyclic ring of biotin and a pocket within each of the four subunits of the proteins. The valeric acid portion is not involved or required for the interaction (Green, 1975 Wilchek and Bayer, 1988). This characteristic allows modification of the valeric acid side chain without affecting the binding potential toward avidin or streptavidin. 

Figure 2.147. Glide s scoring function, which accounts for hydrophobic enclosure and accurately rewards hydrogen bonding interactions, is able to uniquely explain the high affinity with which biotin binds to streptavidin. Here, enclosing hydrophobic groups are colored green, while H-bonding protein residues are renered as tubes...

Streptavidin-biotin interactions are another means of coupling fibrils to a surface, as shown in Figure 14. The binding between streptavidin-biotin is a noncovalent interaction that is remarkably strong and stable. Inoue and... 

Yet another variation of the Western blot exploits the strong interaction between biotin and streptavidin. Biotin is a 244-Da vitamin found in all cells (Fig. 18-4). Streptavidin is a 75-kDa, tetrameric protein isolated from Streptomyces avidinii. Each streptavidin monomer is capable of binding a single molecule of biotin. The dissociation constant of the streptavidin/biotin complex is on the order of 10-1S M, one of the strongest noncovalent interactions found in nature. Many reagents are commercially... 

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