English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins

Kohl, T., Heinze, K. G., Kuhlemann, R., Koltermann, A., & Schwille, P. (2002). A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proceedings of the National Academy of Sciences of the United States of America, 99(19), 12161-12166. Retrieved from http://www.pnas.org/content/99/19/12161.full.pdf+html.

Item is

Basic

show hide
Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-F2F2-7 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-8180-6
Genre: Journal Article

Files

show Files
hide Files
:
599496.pdf (Publisher version), 305KB
Name:
599496.pdf
Description:
-
Visibility:
Public
MIME-Type / Checksum:
application/pdf / [MD5]
Technical Metadata:
Copyright Date:
-
Copyright Info:
-
License:
-

Locators

show

Creators

show
hide
 Creators:
Kohl, T.1, Author              
Heinze, K. G.1, Author              
Kuhlemann, R., Author
Koltermann, A.1, Author              
Schwille, P.1, Author              
Affiliations:
1Research Group of Experimental Biophysics, MPI for biophysical chemistry, Max Planck Society, ou_578554              

Content

show
hide
Free keywords: -
 Abstract: 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.

Details

show
hide
Language(s): eng - English
 Dates: 2002-09-17
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Proceedings of the National Academy of Sciences of the United States of America
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: -
Pages: - Volume / Issue: 99 (19) Sequence Number: - Start / End Page: 12161 - 12166 Identifier: -