Artifacts fluorescence quenching

The complicated nature of the instrumentation required for these experiments may also result in artifacts which may cause the fluorescence quenching to not conform to the model. Fluctuations in the proportioning of solutions A and B in the post-column reagent pump may result in error in the total Cu concentration reported as well as variation in concentration of Cu " in the post-column system throughout an experiment. Likewise, fluctuations in pump flow rate of both the eluent and post-column reagent pumps may also effect the total Cu concentration in the post-column system. Since each experiment was conducted over nine to twelve hours, temperature of the system and the fluorescence detector lamp output may also have contributed to variations in fluorescence quenching. A decreased run time for the chromatographic method and a decrease in the number of sample runs for each experiment may help to reduce this artifact. 

Matrix-isolation Shpol skii fluorimetry has been shown to eliminate quantitative artifacts from quenching and aggregation phenomena occurring in Shpol skii low-temperature solid matrices. The linear dynamic range for the fluorescence of PAHs in vapor-deposited alkane matrices smpasses that of solid-state Shpol skii fluorimetry. Thus, the analytical calibration graph for the fluorescence of benzo[ ]ant-hracene in -heptane is linear from 30 pg (the detection limit) to a maximmn amormt of over 35 pg. The... 

Figure 2. (Top) Stern-Volmer plots for the quenching of the fluorescence of colloi fcl CdS in AOT-entrapped water pools in isooctane by RMV + (0), MV2+ 4Q), and PhSH (0) (Bottom) Absorption and emission spectra of colloidal CdS in AOT entrapped water pools in isooctane. The shoulder observed at 400 nm is due to a spectrometer artifact.

In general, a FRET quench readout is simple. A broad range of available fluorescence donors and acceptors allows cost-efficient operations in an industrialized HTS and automated compound profiling environment. On the other hand, the readout can suffer from inner filter effects due to high absorption coefficients of the dyes and fluorescence artifacts by the tested compounds, resulting in enhanced false positive and false negative rates. Moreover, the readout is limited to substrates in which short distances between donor and acceptor dye can be realized without disturbing the interaction of enzyme and substrate. The flexibility of the peptide conformation makes the prediction of the effective distance between the dyes and consequently the prediction of the FRET effect difficult. The distance between donor and acceptor cannot be easily approximated by the mean hydrodynamic radii of the dyes. 

FRET-based readouts are the most common methods applied for endopeptidase activity assays. In general, the FRET readout is simple, numerous assay examples are published, many fluorescently labeled substrates are commercially available, and labeling procedures are well established. Furthermore, broad range of fluorescence donors and acceptors is available. FRET quench substrates allow fast and cost-efficient operations in an industrialized HTS environment. However, in many cases, the assays suffer from fluorescence artifacts from the tested compounds. This can lead to falsepositive and false-negative results because the most frequently used FRET quench dyes are excited in the ultraviolet range. 

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