Authors: Eggeling, C.; Jager, S.; Winkler, D.; Kask, Peet

Source: Current Pharmaceutical Biotechnology, Volume 6, Number 5, October 2005 , pp. 351-371(21)

Publisher: Bentham Science Publishers

Abstract:

We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

Keywords: fluorescence fluctuation spectroscopy; fluorescence correlation spectroscopy; fluorescence intensity distribution analysis; forster resonance energy transfer; confocal microscopy; protease reaction

Document Type: Review article

DOI: http://dx.doi.org/10.2174/138920105774370571

Affiliations: 1: Max-Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Goettingen, Germany.

Publication date: 2005-10-01

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• Current Pharmaceutical Biotechnology aims to cover all the latest and outstanding developments in Pharmaceutical Biotechnology. Each issue of the journal contains a series of timely in-depth reviews written by leaders in the field covering a range of current topics in both pre-clinical and clinical areas of Pharmaceutical Biotechnology. Current Pharmaceutical Biotechnology is an essential journal for academic, clinical, government and pharmaceutical scientists who wish to be kept informed and up-to-date with the latest and most important developments.

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