Time-resolved Fluorescence Energy Transfer

Horton, R.A. et al. 2007. A substrate for deubiquitinating enzymes based on time-resolved fluorescence energy transfer between terbium and yellow fluorescent protein. Anal. Biochem. 360, 138-143. 

Earnshaw, D. L., Moore, K. J., Greenwood, C. J., Djaballah, H., Jurewicz, A. J., Murray, K. J., and Pope, A. J. Time-resolved fluorescence energy transfer DNA helicase assays for high-throughput screening. /. Biomol. Screening 4 239-248, 1999. 

A study at Novartis compared hits obtained by screening 30,000 compounds for tyrosine kinase activity in three different assays a scintillation proximity assay (SPA), a homogeneous time-resolved fluorescence energy transfer assay (HT-FRET), and a fluorescence polarization (FP) assay.Library compounds were screened as mixtures of five in a well, which meant that after active wells were identified, the five constituents would be deconvoluted by screening each compound individually or using other methods to establish which of the compounds was active. " When this was done SPA, HT-FRET, and FP turned up 30, 59, and 64 hits respectively. A low degree of overlap was observed between the sets. Only four or seven compounds, depending on where the activity bars were set, would have been identified... 

Maurel, D., Kniazeff, J., Mathis, G., Trinquet, E., Pin, J. P. and Ansanay, FI. (2004). Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology. Anal. Biochem. 329, 253-62. 

Time-resolved fluorescence spectroscopy is widely used as a research tool in biochemistry and biophysics. These uses of fluorescence have resulted in extensive knowledge of the structure and dynamics of biological macromolecules. This information has been gained by studies of phenomena that affect the excited state, such as the local environment, quenching processes, and energy transfer. 

Newman, M. and Josiah, S., Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies SRC kinase as a model system, /. Biomol. Screen., 9, 525, 2004. 

Klostermeier, D. and D. P. Millar. Time-resolved fluorescence resonance energy transfer A versatile tool for the analysis of nucleic acids. Biopolymers 61, 159-179 (2002). 

The flnorescence dynamics imaging was carried ont by monitoring time-resolved fluorescence at 530 nm, where the rise of the tetracene fluorescence is observed, at different positions of the microcrystal. The np-converted fluorescence data were recorded every 1 pm distance. To shorten the measnrement time at each point, the up-converted signal was sampled at only fonr delay time points (- 5, 5,15, 30 ps), and then the energy transfer time, x, was evalnated by htting with the following single-exponential fnnction ... 

The ultrafast excitation-energy transfer of the J-aggregates on the octahedral AgBr is studied by the time-resolved fluorescence-anisotropy decay (r(t)) measurements [9]. They are biphasic with two time constants of -0.15 ps and 2-7 ps as shown in Fig. 6. Each phase should reflect some difference in the orientation of the dye molecules of the J-aggregates. 

When deciding to study the dynamics of electronic excitation energy transfer in molecular systems by conventional spectroscopic techniques (in contrast to those based on non-linear properties such as photon echo spectroscopy) one has the choice between time-resolved fluorescence and transient absorption. This choice is not inconsequential because the two techniques do not necessarily monitor the same populations. Fluorescence is a very sensitive technique, in the sense that single photons can be detected. In contrast to transient absorption, it monitors solely excited state populations this is the reason for our choice. But, when dealing with DNA components whose quantum yield is as low as 10-4, [3,30] such experiments are far from trivial. 

Wang, G., J. Yuan, K. Matsumoto, et al. 2001. Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer. Talanta 55 1119-1125. 

The fluorescence lifetime of a fluorophore is highly sensitive to its molecular environment. Many macromolecular events, such as rotational diffusion, resonance-energy transfer, and dynamic quenching, occur on the same timescale as the fluorescence decay. Thus, time-resolved fluorescence spectroscopy can be used to investigate these processes and gain insight into the chemical surroundings of the fluorophore. 

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