Protein Protocols Protein Protocols

Conjugation of Fluorochromes 351
47
Conjugation of Fluorochromes to Antibodies
Su-Yau Mao
1. Introduction
The use of specific antibodies labeled with a fluorescent dye to localize substances
in tissues was first devised by A. H. Coons and his associates. At first, the specific
antibody itself was labeled and applied to the tissue section to identify the antigenic
sites (direct method) (1). Later, the more sensitive and versatile indirect method (2)
was introduced. The primary, unlabeled, antibody is applied to the tissue section, and
the excess is washed off with buffer. A second, labeled antibody from another species,
raised against the IgG of the animal donating the first antibody, is then applied. The
primary antigenic site is thus revealed. A major advantage of the indirect method is
the enhanced sensitivity. In addition, a labeled secondary antibody can be used to locate
any number of primary antibodies raised in the same animal species without the
necessity of labeling each primary antibody.
Four fluorochromes are commonly used: fluorescein, rhodamine, Texas red, and
phycoerythrin (3). They differ in optical properties, such as the intensity and spectral
range of their absorption and fluorescence. Choice of fluorochrome depends on the
particular application. For maximal sensitivity in the binding assays, fluorescein is the
fluorochrome of choice because of its high quantum yield. If the ligand is to be used in
conjunction with fluorescence microscopy, rhodamine coupling is advised, since it has
superior sensitivity in most microscopes and less photobleaching than fluorescein.
Texas red (4) is a red dye with a spectrum that minimally overlaps with that of fluorescein;
therefore, these two dyes are suitable for multicolor applications. Phycoerythrin
is a 240-kDa, highly soluble fluorescent protein derived from cyanobacteria and
eukaryotic algae. Its conjugates are among the most sensitive fluorescent probes available
(5) and are frequently used in flow cytometry and immunoassays (6). In addition,
the newly introduced Alexa fluorochromes are a series of fluorescent dyes with excitation/emission
spectra similar to those of commonly used ones, but are more fluorescent
and more photostable (7).
Thiols and amines are the only two groups commonly found in biomolecules that
can be reliably modified in aqueous solution. Although the thiol group is the easiest
functional group to modify with high selectivity, amines are common targets for modifying
proteins. Virtually all proteins have lysine residues, and most have a free amino
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
351