Biotinylation reagents techniques

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. 

The following sections discuss the main techniques used to make avidin (or streptavidin) conjugates and various biotinylated components. Chapter 8, Section 3, should be consulted for a complete overview of the biotinylation reagents currently available. 

Perhaps the most common method of DNA biotinylation is through enzymatic incorporation with the use of a biotin-labeled deoxynucleoside triphosphate. First reported by Langer et al. and Leary et al. in 1981, the procedure is probably the most popular nonradioactive labeling technique reported for oligonucleotide probes. Although biotinylated derivatives of dCTP and dATP are reported in the literature, by far the most frequently employed derivative is biotin—dUTP prepared from the reaction of an amine-modified dUTP with an amine-reactive biotinylation reagent, such as NHS-LC—biotin (Chapter 8, Section 3.1). 

Scheurer, S.B., Roesli, C., Neri, D. and Elia, G. (2005) A comparison of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques for 2-D peptide mapping of membrane proteins. Proteomics 5, 3035-3039. 

The application of biotinylated receptor substrates is another approach, incubating the labeled substrate with the receptors prior to isolation on an avidin-coated support. In such cases, biotinylation with a cleavable biotinylation reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin would be essential for recovery of the isolated receptor. Alternatively, the receptor could be recovered by substrate competition. Perhaps one of the major drawbacks to the application of affinity techniques is the relative low molecular weight or small size of the receptor substrates, making them difficult ligands to immobilize. However, affinity procedures have been applied to the purification of a number of different receptors although relatively little work has been reported on those involved in the processing of neurotransmittors, neuropeptides, and hormones [1,2]. 

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 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. 

Biotinylated enzymes can be used as detection reagents in avidin—biotin assay procedures. Particularly, in the bridged avidin—biotin (BRAB) approach or the ABC technique (Chapter 13, Section 2), a biotin-labeled enzyme is used as the signaling agent... 

A blueprint for the majority of the following IPCR applications was introduced in 1993 by Zhou et al. [24] with the appositely termed Universal-IPCR protocol. In this technique, the antigen to be detected is coupled subsequently with an antigen-specific primary antibody, a species-specific biotinylated detection antibody, STV, and a biotinylated DNA in four incubation steps, each separated by a washing step for removal of unbound reagents (see Figs. 2B and 3B). 

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