Fluorophore-coupled

It is to be hoped that this chapter will have given readers a better understanding of the basic principles of ion recognition detected by changes in photophysical properties of a fluorophore coupled to an ionophore. Examples have been chosen to illustrate the immense variety of structures that have been already designed and the inexhaustible possibilities of creating new systems. 

Fluorescence staining for flow cytometric analysis falls into three categories methods in which a fluorescent ligand accumulates on or within the cell (see Chapters 36,38) methods that require the ligand to interact with a cellular component to release the fluorophore or result in light emission (see Chapters 34,39) and methods that rely on fluorophore-coupled antibody binding (see Chapters 32,33). 

Fig. 5.2. Methods for detection of passenger-ligand interaction. (A) Fluorophore-coupled ligand (B) fluorophore-coupled antibody (C) quaternary complex generated by subsequent rounds of incubation with ligand, primary antibody, biotinylated second antibody, and strep ta-vidin, R-phycoerythrin conjugate (D) quaternary complex generated by subsequent rounds of incubation wi tli ligand, primary antibody, biotinylated second antibody, and streptavidin-coated magnetobeads. L ligand P passenger.

This technique is used to sense the proximity of two fluorescently labeled molecules. The fluorophore couple is chosen to have a donor that in the excited state transfers its energy by dipole-dipole coupling to an acceptor, that is, the second fluorophore, which re-emits the light at a longer wavelength [117]. Among others, FRET pairs with appropriate spectral overlaps are BFP-GFP or CFP-YFP. Because the transfer efficiency correlates with the inverse sixth power of the distance between the fluorophores and depends on their spatial orientation, the experimental detection of FRET is possible only for pairs separated in space by 1-10 nm. If position and orientation of the fluorophore pair are favorable and ERET occurs, the fluorescent emission of the donor is quenched whereas the acceptor begins to fluoresce. The transfer efficiency can be derived by the ratio of the two emissions [118]. 

Rapid Profiling of IgG A -Glycans by Fluorophore-coupled Oligosaccharide Electrophoresis has the Potential of Differentiating Rheumatic Diseases... 

Frears ER, Merry AH and Axford JS. Rapid profiling of IgG N-glycans by fluorophore-coupled oligosaccharide electrophoresis has the potential of differentiating rheumatic diseases. Arth Rheum 1998 41(9) Suppl. S85 No. 317,... 

By using phase sensitive detection, the detector phase angle can be adjusted to be exactly out of phase with the phase-delayed emission from any single fluorophore, suppressing its contribution to the total emission signal. Phase sensitive detection, coupled with... 

The data were collected using fluorescence measurements, which allow both identification and quantitation of the fluorophore in solvent extraction. Important experimental considerations such as solvent choice, temperature, and concentrations of the modifier and the analytes are discussed. The utility of this method as a means of simplifying complex PAH mixtures is also evaluated. In addition, the coupling of cyclodextrin-modified solvent extraction with luminescence measurements for qualitative evaluation of components in mixtures will be discussed briefly. 

Jackson, P., The use of polyacrylamide-gel electrophoresis for the high-reso-lution separation of reducing saccharides labeled with the fluorophore 8-ami-nonaphthalene-l,3,6-trisulfonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device, Biochem. ]., 270, 705, 1990. 

Three types of reactive spectrally distinct fluorophores, namely lissamine rhoda-mine (LR) 80, 7-dimethylaminocoumarin (DMAC) 81, and bodipy-630 (BDPY) 82 dyes, prepared by coupling 3-azidopropylamine or propargylamine to commercially available amine-reactive dyes were evaluated for the use in selective dye-labeling of newly synthesised proteins in Rat-1 fibroblasts. 

For instance, a dendrimer easily can be coupled with a large number of fluorescent dyes and still provide additional coupling sites for biotinylation. The only limitation to the number of fluorescent modifications is if fluorescence quenching starts to take place, in which case no further modifications will result in increased signal. A series of such conjugates using different levels of fluorophore modification should be done to determine the optimal level of dye-to-dendrimer before quenching occurs. 

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