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          Institute: MPI für biophysikalische Chemie     Collection: Abteilungsunabhängige Arbeitsgruppen     Display Documents



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ID: 16661.0, MPI für biophysikalische Chemie / Abteilungsunabhängige Arbeitsgruppen
A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins
Authors:Kohl, T.; Heinze, K. G.; Kuhlemann, R.; Koltermann, A.; Schwille, P.
Language:English
Date of Publication (YYYY-MM-DD):2002-09-17
Title of Journal:Proceedings of the National Academy of Sciences of the United States of America
Volume:99
Issue / Number:19
Start Page:12161
End Page:12166
Review Status:Peer-review
Audience:Not Specified
Abstract / Description:GFP and the red fluorescent protein, DsRed, have been combined to design a protease assay that allows not only for fluorescence resonance energy transfer (FRET) studies but also for dual-color crosscorrelation analysis, a single-molecule- based method that selectively probes the concomitant movement of two distinct tags. The measurement principle is based on a spectrally resolved detection of single molecules diffusing in and out of a diffraction-limited laser focus. Double-labeled substrate molecules are separated into two single-labeled products by specific cleavage at a protease cleavage site between the two flanking tags, DsRed and GFP, thus disrupting joint fluctuations in the two detection channels and terminating FRET between the two labels. In contrast to enzyme assays based solely on FRET, this method of dual-color crosscorrelation is not limited to a certain range of distances between the fluorophores and is much more versatile with respect to possible substrate design. To simplify the measurement setup, two-photon excitation was used, allowing for simultaneous excitation of both tags with a single infrared laser wavelength. The general concept was experimentally verified with a GFP-peptide-DsRed construct containing the cleavage site for tobacco etch virus protease. Two-photon excitation in the infrared and the use of cloneable tags make this assay easily adaptable to intracellular applications. Moreover, the combination of FRET and crosscorrelation analysis in a sing le-molecule-based approach promises exciting perspectives for miniaturized high-throughput screening based on fluorescence spectroscopy.
Comment of the Author/Creator:Date: 2002, SEP 17
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für biophysikalische Chemie/AG Petra Schwille
External Affiliations:DIREVO Biotech AG, D-50829 Cologne, Germany
Identifiers:URL:http://www.pnas.org/content/99/19/12161.full.pdf+h...
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