Time-domain fluorescent plate reader for cell based protein-protein interaction and protein conformation assays

Fluorescence lifetime measurement is widely used in the biological sciences due to its inherent sensitivity and concentration independence. Frequency domain high-throughput plate readers and time-resolved energy transfer (TRET) plate readers are in common use and have been successful in a variety of...

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Bibliographic Details
Published in:	Journal of Biomedical Optics 2006-09, Vol.11 (5), p.054024-0540210
Main Authors:	Jones, Phill B, Herl, Lauren, Berezovska, Oksana, Kumar, Anand T. N, Bacskai, Brian J, Hyman, Bradley T
Format:	Article
Language:	eng
Subjects:	Algorithms Equipment Design Equipment Failure Analysis Fluorescence Resonance Energy Transfer - instrumentation Fluorescence Resonance Energy Transfer - methods fluorescence spectroscopy molecular spectroscopy Protein Conformation Protein Interaction Mapping - instrumentation Protein Interaction Mapping - methods Proteins - chemistry Proteins - metabolism Proteins - ultrastructure Reproducibility of Results Sensitivity and Specificity
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Summary:	Fluorescence lifetime measurement is widely used in the biological sciences due to its inherent sensitivity and concentration independence. Frequency domain high-throughput plate readers and time-resolved energy transfer (TRET) plate readers are in common use and have been successful in a variety of applications ranging from basic biochemistry to drug discovery. Time-domain systems would have advantages due to their ability to distinguish both FRETing and non-FRETing populations, but have been difficult to develop due to inherent difficulties with background autofluorescence and lifetime component separation. Using a modified commercial lifetime plate reader, we demonstrate a method for removal of the complex auto-fluorescent background decay, described using a stretched exponential function (StrEF). We develop a generalized multi-exponential fitting algorithm (GeMEF), which progressively accounts for confounding lifetime components in FRET-based assays using a series of control experiments. We demonstrate the separability of FRET strength and efficiency and apply the technique to protein-protein interactions and protein conformational assays in a cell-based format. Presenilin 1 (PS1) is known to be important in Amyloid Precursor Protein (APP) processing in Alzheimer's disease. Using transfected cells, we demonstrate APP-PS1 interactions by FRET in a cell-based, 96-well plate format.
ISSN:	1083-3668 1560-2281