Indirect Quenching Fluorescence

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

The existence of a stable electron acceptor in PSII has long been recognized indirectly by observing the fluorescence increase of chlorophyll a occurring upon illumination. This increase was attributed to the reduction of an acceptor and the consequent closing of a PSII-RC. The acceptor was defined as Q (for quencher) and proposed to quench fluorescence when oxidized [83]. 

Indirect quenching FIA, also called fluorescence protection immvmoassay, is based on the use of fluorescence-labeled antigens and antibodies directed against the label. When antilabel antibodies bind to the labeled probe, the fluorescence is almost completely quenched. This approach can be either... 

However, the direct determination of absorption at the wavelength of maximum absorption is more sensitive (or in the worst case at least as sensitive) as the indirect measurement of absorption by fluorescence or phosphorescence quenching. 

Indirect evidence for the formation of a complex intermediate in diffusional energy transfer processes may be provided by the measurement of kt from both donor quenching and acceptor sensitization at low temperatures where kQC kf = 1/T° in this case exciplex relaxation should reduce the quencher sensitization efficiency but leave the donor fluorescence quenching constant unchanged. 

The MEKC separation of explosives was achieved, except that three isomers of nitrotoluenes cannot be resolved [620]. A peak height RSD was 1.7-3.8% for TNB, DNB, TNT, tetryl, 2,4-DNT, 2,6-DNT, and 2-amino-4,6-DNT. But the linear ranges for TNB, DNB, TNT, and tetryl were only 1-5 ppm This narrow linear range, which is caused by the indirect LIF detection based on fluorescent quenching, may be sufficient for screening, but is certainly not useful for quantitation [620]. 

A representative example of indirect imaging with SWNTs labeled with fluorescent molecules was illuminated by Dai and coworkers.102 They solubilized as-produced SWNTs via sonication with fluorescein-modified poly (ethylene glycol) (Fluor-PEG, 114 PEG units). The absorption maximum of the nanotube-attached Fluor-PEG was red shifted 3 nm from that of free fluorescein, and the fluorescence intensity was quenched about 67% in phosphate buffer solution (pH 7.4). In the bioevaluation with BT474 breast cancer cells, substantial intracellular fluorescence was observed, suggesting cellular uptake of the Fluor-PEG-functionalized SWNTs.102... 

Figure 6. Time course of change in catalytic specificity (upper panel) and Ca2+ dissociation from extracytoplasmic low affinity sites (lower panel) following phosphorylation of the SR Ca2+-ATPase with ATP. The amount of ADP-insensitive phosphoen-zyme (E2P) was measured in two ways (I) [y-32P]ATP was included in the reaction mixture and the radioactivity incorporated into the enzyme was determined after acid quenching at various time intervals. To remove the ADP-sensitive phosphoenzyme so that only ADP-insensitive phosphoenzyme was measured, ADP was added 4 sec before the quench (upper panel, right scale) (2) by the enhancement of fluorescence from a trinitrophenyl-derivative of ADP bound in the catalytic site in exchange with ADP after the phosphorylation (upper panel, left scale). The change in Ca2+ binding was measured indirectly by use of murexide as an indicator of free Ca2+ in the medium. The data show that Ca2+ dissociates simultaneously with formation of E2P. The data points were taken from Andersen et al., 1985.

Fluorescence Protection Immunoassay (Indirect Fluorescence Quenching... 

Investigation of thermostable protein dynamics by indirect methods such as the kinetics of proteolysis and H-D exchange as well as buried chromophore fluorescence quenching, has led to the conclusion that at ambient temperature their globules are essentially less flexible than for non-thermostable proteins (Vetriani et al., 1998 and references therein). 

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