Avidin conjugation

Patterning of enzyme monolayers on a solid surface was carried out by photoactivation of immobilized monolayer of caged -biotin derivatives in selected areas. Specific oriented binding of enzyme-avidin conjugates could be readily made to the photoactivated zones [42]. Oriented immobilization of G-protein-coupled receptors on a solid surface was also made possible on a biotinylated surface by first immobilizing streptavidin, followed by the immobilization of biotinylated G-protein-coupled receptor [43]. 

Figure 22.21 Antibodies may be conjugated to liposomes using an indirect approach incorporating a (strept)avidin-biotin system. Biotinylated liposomes may be complexed with biotinylated antibodies using (strept)avidin as a bridging molecule or may be complexed with an antibody-(strept)avidin conjugate.

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. 

When photobiotin is irradiated in the presence of DNA the reaction process nonselectively couples a biotin label to every 100-200 base residues. The result is an oligonucleotide probe detectable by the use of (strept)avidin conjugates. The uses of photobiotin for DNA or RNA modification are summarized in Chapter 11, Section 4. 

Bayer, E.A., Skutelsky, E., Wynne, D., and Wilchek, M. (1976) Preparation of ferritin-avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry./. Histochem. Cytochem. 24, 933-939. 

Horikawa, K., and Armstrong, W.E. (1988) A versatile means of intracellular labeling injection of biocy-tin and its detection with avidin conjugates./. Neurosci. Meth. 25, 1-11. 

Incubate sections for 30 min with (strept)avidin conjugated with a fluorophore or with any enzyme (peroxidase or alkaline phosphatase). For localization of low-density antigens (<10K molecules/cell), VECTASTAIN ABC reagent (http //www.vectorlabs.com/) is preferable. 

Visualization of biotin biotin can be visualized using (strept)avidin conjugated with a fluorophore of with an enzyme label. 

Mostly bovine serum albumin (BSA) is used. Gelatin from cold fish, inactivated calf serum, casein hydrolysate, non-fat dry mUk, polyvinylpyrrolidone, or Tween 20 are also suitable. Concentrations from 0.1 to 0.5% (w/v) are sufficient. Casein and mfik are not of first choice if (strept)avidin conjugates are used. 

Holle L, Song W, et al. A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo. Int J Oncol 2003 22 93-98. 

Avidin conjugates of a wide range of fluorophores, phycobiliprotems, secondary antibodies, microspheres, ferritin, and enzymes commonly used in lmmunochemistry are available at reasonable prices, making their small scale preparation impractical and not cost effective (see Note 1). However, conjugations of avidin to specific antibodies, to uncommon enzymes, and to other proteins and peptides are often performed on-site. A general protocol for the conjugation of avidin to enzymes, antibodies, and other proteins is described in this chapter. 

Avidin conjugates of oligodeoxynucleotides are hybrid molecules that not only provide multiple biotin binding sites, but can also be targeted to complimentary DNA or RNA sequences, by annealing interactions. Such conjugates are useful for the construction of macromolecular assemblies with a wide vari-... 

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