Biotinylation techniques

The following sections discuss the concept and use of the avidin-biotin interaction in bioconjugate techniques. Preparation of biotinylated molecules and avidin conjugates also are reviewed with suggested protocols. For a discussion of the major biotinylation reagents, see Chapter 8, Section 3. 

In addition to preparing the avidin or streptavidin conjugates necessary to develop avidin—biotin-based systems, the process of modifying targeting molecules with a 

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The following sections describe the principal reagents available for producing tagged molecules using fluorescent probes, radiolabels, and biotinylation techniques. In many cases, suggested protocols are included (or other sections referenced) for the use of these probes. 

PURPOSE AND RATIONALE This test uses the ex vivo model of comeal organ culture previously discussed combined with surface biotinylation technique. The comeal epithelium functions as a barrier that separates the internal ocular tissues from the external environment and is therefore vulnerable to chemical insult (Gipson and Sugrue 1994). The joint assessment of comeal organ culture and surface biotinylation is a measure of ocular toxicity that is intended to reveal disrupted tight junction barriers following chemical exposure. 

One of the biggest advantages of this culture system is the accessibility of either the apical or basolateral surface to any reagent that can be added to the medium in contact with either of the two surfaces. This is the basis of the biotinylation techniques that have been developed to selectively label proteins present in the apical or the basolateral domains of the plasma membrane of filter-grown cells. 

Lebaron, P. Catala, P. Fajon, C. Joux, F. Baudart, J. Bernard, L. A new sensitive, whole-cell hybridization technique for detection of bacteria involving a biotinylated oligonucleotide probe targeting rRNA and tyramide signal amplification. Appl. Environ. Microbiol. 1997, 63, 3274-3278. 

NHS-LC-biotin can be used to add a biotin tag to monoclonal antibodies directed at certain tumor antigens. The biotinylated monoclonals are allowed to bind to the tumor cell surfaces in vivo, and subsequent administration of an avidin or streptavidin conjugate can form the basis for inducing cytotoxic effects or creating traceable complexes for use in imaging techniques (Hnatowich et al., 1987). 

Purify the biotinylated protein or molecule using dialysis or gel filtration. For small molecule biotinylation where these separation methods may not be appropriate, other procedures may have to be developed, such as reverse-phase chromatography or organic precipitation techniques. 

Colloidal gold-labeled (strept)avidin can be used as highly sensitive detection reagents for microscopy techniques (Cubie and Norval, 1989) (Chapter 24). Finally, cytotoxic substances coupled to (strept)avidin can be used to direct cell-killing activity toward a tumor-cell-bound, biotinylated monoclonal antibody (or other targeting molecule) for cancer therapy (Hashimoto et al, 1984) (Chapter 21). 

The following sections discuss the main techniques used to make (strept)avidin conjugates and various biotinylated components. Chapter 11 and Chapter 18, Section 3 should be consulted for a complete overview of biotinylation reagents. 

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