Avidin, streptavidin

First entry on each line is the tag. Square brackets enclose the probe donor + transfer enzyme (where applicable) or reaction. Probe organic fluorophores, nanoparticles (can be QDs as in methods 2, 3, 8, 9) or a bridging/recognition moiety. If desired, the latter can serve in a second orthogonal reaction ( piggyback strategy). See text. Biotin readout probes linked to avidin, streptavidin, anti-biotin 7[101] ... 

Biotin modification reagents are widely used to attach a biotin group to proteins or other molecules for subsequent use in avidin, streptavidin, or NeutrAvidin separations or assays. Traditional biotin compounds containing aliphatic or other hydrophobic linker arms are discussed in detail in Chapter 11. In this section, the biotin-PEG compounds exclusively are discussed due to their unique hydrophilic properties, which include low nonspecific binding character and low immunogenicity. 

Strepavidin A 60 kD extracellular protein of Streptomyces avidinii with four high-affinity biotin binding sites. Unlike avidin, streptavidin has a near neutral isoelectric point and is free of carbohydrate side chains. 

The strong bond between biotin and avidin/streptavidin can only be released by extreme and often denaturating conditions. For a reversible binding based on this complex, o-desthiobiotin can be used instead of biotin (see Fig. 13). This conjugate can be separated at room temperature by exposure to an excess amount of biotin, respectively desthiobiotin [42]. 

Streptavidin is another biotin binding protein isolated from Streptomyces avidinii that can overcome some of the nonspecificities of avidin (Chaiet and Wolf, 1964). Similar to avidin, streptavidin contains four subunits, each with a single biotin binding site. After some postsecretory modifications, the intact tetrameric protein has a molecular mass of about 60,000 D, slightly less than that of avidin (Bayer et al., 1986, 1989). 

Other molecules can be used in this type of assay approach. Hydrazide-modified avidin, streptavidin, lectins, biocytin, fluorescent probes, and other detectable molecules can specifically detect glycoconjugates in this manner (Wilchek and Bayer, 1987). 

Because avidin is a glycoprotein and has an isoelectric point (pi) of 10, it has a propensity to bind non-specifically to lectin-like and negatively charged tissue components at physiological pH. In contrast to avidin, streptavidin has a more neutral isoelectric point and lacks the carbohydrate moieties. These differences result in less nonspecific tissue binding. 

Of primary importance for developing a high-performance DNA chip is to introduce a functional probe DNA to the substrate surface and to avoid nonspecific adsorption of the target sample. Methods for DNA immobilization can be divided to physical, biotin-avidin/streptavidin (SA) and chemical techniques including self-assembly via thiolated-linker immobilization (Fig. 2). 

A closely similar protein, streptavidin, has been isolated from culture filtrates of several species of Streptomyces. Unlike avidin, streptavidin is not glycosylated and has an acidic isoelectric point. It binds biotin with a similarly high affinity. 

Biotinylated probes can be detected with avidin, streptavidin or modified avidin (Yehle et al., 1987). Replacing phosphate-buffered saline by SSC during hybridization reduces the background of unmodified avidin (fluoresceinated) making it superior to modified avidin for single-copy detection (McNeil et al., 1991). 

Avidin/streptavidin biotin. Sensitive. Laborious. Toxic chromogens. Need to block endogenous biotin activity. 

Key words Biotinylation, Antibody, Immunoglobulin, IgG, Biotin, Avidin, Streptavidin, NHS-biotin, sulfo-NHS-biotin... 

Key words Colloidal gold, Avidin, Streptavidin, Biotin, Electron microscopy, Postembedding labeling... 

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