Protein conjugates, fluorescence

Most of the molecules introduced in this chapter are hydrophobic. Even those molecules that have been functionalized to improve water-solubility (for example, CCVJ and CCVJ triethyleneglycol ester 43, Fig. 14) contain large hydrophobic structures. In aqueous solutions that contain proteins or other macromolecules with hydrophobic regions, molecular rotors are attracted to these pockets and bind to the proteins. Noncovalent attraction to hydrophobic pockets is associated with restricted intramolecular rotation and consequently increased quantum yield. In this respect, molecular rotors are superior protein probes, because they do not only indicate the presence of proteins (similar to antibody-conjugated fluorescent markers), but they also report a constricted environment and can therefore be used to probe protein structure and assembly. 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

Figure 14.4. Diagram of three basic immunoassay formats, (a) Common competitive format (b) competitive assay with immobilized antigens bound to a carrier protein (immunometric assay) (c) two-site immunometric assays. Haptenic analytes are indicated as triangles, whereas larger-molecular-weight analytes are shown as teardrop shapes. Conjugated fluorescent probes are denoted by the letter "F. ...

Table 14.2. Fluorescence Lifetime Distributions of Some Fluorophore-Protein Conjugates...

Giloh, H. and Sedat, M. (1982) Fluorescence microscopy reduced photobleaching of rhodamine and fluorescein protein conjugates by A-propyl gallate. Science 217, 1252-1255. 

If the ligand protein is fluorescently labeled, resuspend the cells in 10 pL PBS and subject directly to FACS. If the ligand protein is biotinylated (protocol 2), resuspend the cells in 10 pL PBS containing streptavidin, R-phycoerythrin conjugate (1 10 dilution in PBS). Incubate on ice again for 10 min. 

Steiner, R. F., and H. Edelhoch Fluorescent protein conjugates. Chem. 

The utihzation of fluorescence dyes for analytical measurements enhances the sensitivity for the detection of the molecules of interest. First, Cronick and Little made use of evanescent wave excitation for a fluorescence immunoassay, in 1975. By using totally internally reflected light, they excited the fluorescence of a fluorescein-labeled antibody which has become bound to a hapten-protein conjugate adsorbed on a quartz-plate in an antibody solution [41]. Contrary to the label-free high-refractive-index sensors where the mass of the molecule of interest is... 

Acrolein-containing latex particles were used for immobilization of proteins. The latex particles were prepared by emulsifier-free polymerization of acrolein and styrene (Table I). Fluorescent latex particles were prepared by mixing fluorescent dyes such as Hostalux KCB (Hoechst) or coumarin-6 into styrene before the emulsion polymerization (Table I). To increase the immunological sensitivity of the protein-conjugated latex particles, a hexyl group was introduced between the latex particle and the protein (Protein-spacer-latex). Proteins such as human serum albumin (HSA), anti-human serum albumin (anti-HSA-IgG), and a fragmented antibody (anti-HSA-... 

Fluorescent cholera toxin Fluorescent dextran conjugates Fluorescent Dil-LDL Fluorescent fusion proteins (GFP) Fluorescent / neutralizing IgG Fluorescent transferrin Hypertonic sucrose Ikarugamycin... 

Dandliker, W. B., Portmann, A. J. Fluorescent Protein Conjugates. In Excited States of Proteins and Nucleic Acids. Steiner, R. F., Weinigh, I. (Eds.) p. 199, New York Plenum 1971... 

As outlined in this chapter, ARs can be visualized using fluorescent antibodies (9,17,42), protein conjugates (1,3,4,8), or ligands (33,35). Each technique has its own set of advantages and disadvantages. However, all present similar issues for effective visualization and quantification. 

Barak LS, Ferguson SSG, Zhang J, Martenson C, Meyer T, Caron MG. Internal trafficking and surface mobility of a functionally intact P2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol 1997 51 177-184. 

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