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Luminescence Resonance Energy Transfer Analysis of RNA Polymerase Complexes

Luminescence Resonance Energy Transfer Analysis of RNA Polymerase Complexes,10.1006/meth.2001.1214,Methods,Tomasz Heyduk

Luminescence Resonance Energy Transfer Analysis of RNA Polymerase Complexes   (Citations: 9)
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Resonance energy transfer allows measurement of atomic-scale distances under a variety of solution conditions. Luminescence resonance energy transfer (LRET) is a variant of energy transfer measurement in which lanthanide chelates are used as the probes. The unusual properties of lanthanide emission, in particular their long microsecond-scale lifetimes, offer several advantages for energy transfer measurements with biological samples. One of the unique features of LRET is the ability to measure energy transfer under conditions where severe heterogeneity of labeled macromolecules exists. This feature of LRET is the special emphasis of this article. We describe here LRET methodology with a particular attention to using sensitized acceptor emission to determine efficiency of energy transfer. Although we employed this technique in the characterization of Escherichia coli RNA polymerase complexes it is readily compatible with the study of essentially any protein.
Journal: Methods , vol. 25, no. 1, pp. 44-53, 2001
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    • ... The fact that the resonance energy transfer is distance dependent makes FRET and LRET highly suitable for the detection and quantification of protein-protein interactions. The key difference between FRET and LRET is that LRET utilizes a lanthanide chelate as the donor instead of a typical organic fluorophore. The lanthanide chelate's emission is technically not fluorescence (i.e. arising from a singlet to singlet transition) and has a very long fluorescent half-live (∼ms) compared to the short half-life (∼ns) of most organic fluorophores. The longer half-life of the lanthanide chelates provides the opportunity to time-gate or delay a measurement after excitation, allowing for reduction of background fluorescence. For a more detailed explanation of LRET, see the following publications ...

    Bryan T. Glaseret al. Studying the Salt Dependence of the Binding of sigma70 and sigma32 to ...

    • ...The details of LRET have been elegantly described in recent reviews by Selvin (8) and Heyduk (16) and will be covered only briefly here...

    Veit Bergendahlet al. Luminescence Resonance Energy Transfer-Based High-Throughput Screening...

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