Antibody Conjugates for Cell Biology

Antibody conjugates are extremely useful reagents for probing many biologically and chemically important molecules in vitro or in vivo. This unit presents some basic protocols for conjugating antibodies with fluorescent dyes, with biotin (see Basic Protocol 1), and with enzymes (see Basic Protocol 2). These protocols have been selected because they are relatively easy to perform and have high rates of success. The basic approach for conjugating antibodies is to derivatize amino groups on the antibody with the dye or biotin derivative of choice. Conjugation at amine sites is simple to perform, and, with a fluorescent dye, it generally yields brightly fluorescent conjugates. The conjugates retain high levels of biological activity, notwithstanding the fact that the labels react with random amines on the antibody molecule, and they may even react near or at the antigen-binding site. Labeling of the amines is the most widely used commercial method to tag antibodies. In this unit, suggestions are presented for variations on these basic protocols that are useful when quantities of available antibody are limited. Methods for estimating antibody concentration are also included (see Support Protocol).