To exploit the extraordinary specificity and high affinity of biotin binding to biotin-binding proteins like streptavidin, avidin, and their derivatives, researchers often need to conjugate biotin to one or more of their reaction components in vitro (biotinylation). Such biotinylated molecules can then serve as targets in many detection protocols using biotin-binding proteins or antibiotin antibodies labeled with fluorescent, enzyme, radioactive, or other reporter groups. Also, biotinylated molecules can be isolated and purified based on their highly specific, predictable, and with appropriate reagents, reversible interactions with immobilized biotin-binding proteins. Researchers have a wide range of options available for such affinity-based applications. The reader is referred to refs. 1-8 for detailed explanations of these subject areas.
Virtually any type of biomolecule can be biotinylated using a variety of reactive biotin derivatives that react covalently with specific molecular targets. These include, but are not limited to: 1) succinimidyl esters of biotin that label primary amines found on protein lysine residues and at the N-terminus; 2) biotin maleimides and other derivatives that react with endogenous or introduced thiol groups; 3) biotin hydrazides that label carbohydrate moieties under appropriate conditions; and 4) photoreactive biotin compounds that label biomolecules nonspecifically in the presence of light at the appropriate wavelength. Proteins, nucleic acids, carbohydrates, lip-ids, and conjugates thereof can be efficiently biotinylated in vitro with these reagents. Biotinylation of whole cells is also possible with some of these compounds (e.g., FluoReporter® Cell Surface Biotinylation Kit, Molecular Probes, Inc., Eugene, OR).
This chapter describes optimized protocols for conjugating antibodies to biotin using representative examples of the reagents mentioned previously. We will emphasize covalent biotinylation of polyclonal and monoclonal IgG molecules, but other types of im-munoglobulins as well as immunoglobulin fragments can be biotinylated using the methods described here. Covalent biotinylation of antibodies on their primary amines is the most popular and simplest method, so we will emphasize this approach. Detailed explanations of the many available chemical biotinylation reagents, the chemistry of their reactive groups, and the huge number of biotinylated antibody-based applications are beyond the present scope, so the reader is referred to refs. 1-8 for further information.
However, researchers should be aware that a new noncovalent method for biotinylating antibodies has recently become available. Zenon™ technology from Molecular Probes, Inc., is a unique immunolabeling system that enables researchers to attach biotin, desthiobiotin, and other tags to mouse, rabbit, and human antibodies on their respective Fc regions. Zenon technology requires the use of anti-Fc F(ab) fragments as carriers for biotin, desthiobiotin, or many other reporter molecules . It is designed to rapidly biotinylate or otherwise label submicrogram amounts of poly- and monoclonal antibodies, and unlike the covalent biotinylation methods described here, the antibody targets can be labeled in ascites fluids or in the presence of extraneous proteins like bovine serum albumin or gelatin. Extensive descriptions and numerous applications of Zenon technology are available at www.probes.com/Zenon on the Internet.
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