Resonance energy transfer limitations

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

Abbreviations AOD, Acousto-optical deflection BCB, bisbenzyocyclobutadiene CCD, indirect contact conductivity detection CL, chemiluminescence ECD, electron capture detector FCS, fluorescence correlation spectroscopy FRET, fluorescence resonance energy transfer ICCD, integrated contact conductivity detection GMR, giant magnetoresistive LED-CFD, light emitting diode confocal fluorescence detector LIF, laser-induced fluorescence LOD, limit of detection MALDI, matrix-assisted laser desorption ionization PDMS, poly(dimethylsiloxane) PMMA, poly(methylmetha-crylate) SPR, surface plasmon resonance SVD, sinusoidal voltammetric detection TLS, thermal lens spectroscopy. 

This is due to the uniform initial condition k(t), which starts from its maximal value ki) Wrrd3r and drops with time, approaching the stationary limit from above. Contrary to this famous result, the experimental study of delayed fluorescence of anthracene in viscous solution [262] showed quite the opposite time behavior of k t). As it is initially much less than ko, the rate constant increases with time, approaching the long-time asymptote (3.56) from below. The authors of Ref. 262 called this anomaly the anti-Smoluchowski time behavior of the delayed fluorescence. They properly attributed it to a nonuniform distribution of triplets generated by the intersystem conversion from singlets that are preliminary quenched by the resonant energy transfer. 

Second, we present a Forster resonance energy transfer (FRET) imaging method, which has been used to monitor GPGR-mediated dissociation (activation) and reassociation (deactivation) of heterotrimeric G-protein in single live cells (10, 11). Protein/protein interactions cannot be measured by colocalization of the proteins because the limit of resolution of the light microscope using standard techniques is on the order of hundreds of nanometers, and one cannot be certain that two proteins of interest physically interact even... 

The first obvious use of FRET is in sandwich immunoassay. A first report along this line was presented by Ullman et al. in 1976 [168]. Wei et al. investigated 10 combinatorial pairs of conventional fluorescent dyes and assessed the optimal conditions for energy transfer [169]. Oswald et al. described a fluorescence resonance energy transfer immunoassay for human serun albumin (HSA) based on the indo-lium dye Sq635 (Fig. 14.10) and the benz[e]indolium dye Sq660 [83]. A detection limit of 10 M of HSA was reported. 

Bioluminescence and Bioluminescence Resonance Energy Transfer Assays in Mkrofluidks, Fig. 6 (a) Time-dependent BRET ratio of the control assay and the positive (27 pM) assay, (b) Dose response curve for thrombin assay a limit of detection of 19.8 pM and a sensitivity of... 

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