This chapter is devoted to our biotinylation, desthiobiotinylation and haptenylation reagents (Biotinylation and Haptenylation Reagents—Section 4.2) and our biotin and desthiobiotin (DSB-X biotin) conjugates (Biotin and Desthiobiotin Conjugates—Section 4.3). For the detection of biotin and hapten conjugates, we prepare a large assortment of labeled avidin and antibody probes, which are described in Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6 and Anti-Dye and Anti-Hapten Antibodies—Section 7.4, respectively. Our avidin- and biotin-coated FluoSpheres microspheres (Microspheres—Section 6.5) and Qdot nanocrystals (Qdot Nanocrystals—Section 6.6) provide alternative detection technologies that offer a combination of fluorescence intensity and photostability far superior to that of any simple dye conjugate.
The high affinity and specificity of avidin–biotin and antibody–hapten interactions have been exploited for diverse applications in immunology, histochemistry, in situ hybridization, affinity chromatography and many other areas. Biotinylation (Biotinylation and desthiobiotinylation reagents—Table 4.1) and haptenylation (Selected haptenylation reagents and their anti-hapten antibodies—Table 4.2) reagents provide the "tag" that transforms poorly detectable molecules into probes that can be recognized by a labeled detection reagent or an affinity-capture matrix. Once tagged with biotin or a hapten, a molecule of interest—such as an antibody, drug, oligonucleotide, polysaccharide or receptor ligand—can be used to probe cells and tissues, as well as protein and nucleic acid blots and arrays. After finding its target, this tagged molecule can be detected with the appropriate avidin or anti-hapten antibody conjugate labeled with a fluorophore, fluorescent microsphere, enzyme, magnetic particle or colloidal gold. Biotinylated molecules can also be captured with various forms of immobilized streptavidin, such as streptavidin agarose (S951), CaptAvidin agarose (C21386) or streptavidin-coupled magnetic Dynabeads (www.invitrogen.com/handbook/dynabeads). Biotinylated probes can be developed for electron microscopy with NANOGOLD or Alexa Fluor FluoroNanogold streptavidin (N24918, A24926, A24927) or streptavidin-coupled Qdot nanocrystals (Qdot Nanocrystals—Section 6.6). Our extensive array of avidin and streptavidin conjugates are described in Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6.
Detection Methods Compatible with Avidin–Biotin Techniques
Avidin–biotin and antibody–hapten techniques are compatible with flow cytometry and light, electron and fluorescence microscopy, as well as with solution-based methods such as enzyme-linked immunosorbent assays (ELISAs). Moreover, avidin–biotin and antibody–hapten techniques are frequently combined for simultaneous, multicolor detection of multiple targets in complex tissue samples. By judicious choice of detection reagents and sandwich protocols, these techniques can be employed to amplify signals from low-abundance analytes. For example, the bridging method is a common immunohistochemical technique for signal amplification and improved tissue penetration in which avidin or streptavidin serves as a bridge between two biotinylated molecules. Other amplification strategies include the tyramide signal amplification (TSA) technology (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2).
Endogenous Biotin and Biotinidase
Mammalian cells and tissues contain biotin-dependent carboxylases, which are required for a variety of metabolic functions. These biotin-containing enzymes often produce substantial background signals when biotin–avidin or biotin–streptavidin detection systems are used to identify cellular targets (, ). Endogenous biotin is particularly prevalent in mitochondria and in kidney, liver and brain tissues. The reagents in the Endogenous Biotin-Blocking Kit (E21390), which is described in Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, can be used to minimize interference from endogenous biotin in these techniques. In mammalian serum and plasma, biotinylated proteins are susceptible to cleavage by endogenous biotinidases, producing free biotin and unlabeled protein.
CaptAvidin Biotin-Binding Protein
Although binding of biotin to native avidin or streptavidin is essentially irreversible, appropriately modified avidins can bind biotinylated probes reversibly, making them valuable reagents for isolating and purifying biotinylated molecules from complex mixtures. In the CaptAvidin biotin-binding protein (C21385, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6), selective nitration of tyrosine residues in the four biotin-binding sites of avidin considerably reduces the affinity of this protein for biotinylated molecules above pH 9. Consequently, biotinylated probes can be adsorbed to CaptAvidin biotin-binding protein at neutral pH or below and released at ~pH 10. CaptAvidin agarose (C21386, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) is particularly useful for separating and purifying biotin conjugates from complex mixtures.
In contrast to the modified avidin of the CaptAvidin products, our DSB-X biotin technology employs a modified biotin to provide a means of labeling and separating biomolecules, including live cells, under extremely gentle conditions. The DSB-X biotin reagents, which are derivatives of desthiobiotin (Figure 4.1.1) with an additional seven-atom 'X' spacer, have moderate affinity for avidin and streptavidin that is rapidly reversed by low concentrations of free biotin or desthiobiotin at neutral pH and room temperature; the Kd for DSB binding to streptavidin has been reported to be 1.9 nM. The DSB-X Biotin Protein Labeling Kit (D20655, Biotinylation and Haptenylation Reagents—Section 4.2) provides a convenient method for labeling proteins with the amine-reactive succinimidyl ester of DSB-X biotin and purifying the conjugate.
Figure 4.1.1 Comparison of the structures of D-biotin (top) and D-desthiobiotin (bottom).