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Journal of Biomedical Optics

Time-domain fluorescent plate reader for cell based protein-protein interaction and protein conformation assays
Author(s): Phill B. Jones; Lauren Herl; Oksana Berezovska; Anand T. N. Kumar; Brian J. Bacskai; Bradley T. Hyman
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Paper Abstract

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.

Paper Details

Date Published: 1 September 2006
PDF: 10 pages
J. Biomed. Opt. 11(5) 054024 doi: 10.1117/1.2363367
Published in: Journal of Biomedical Optics Volume 11, Issue 5
Show Author Affiliations
Phill B. Jones, Massachusetts General Hospital (United States)
Lauren Herl, Massachusetts General Hospital (United States)
Oksana Berezovska, Massachusetts General Hospital (United States)
Anand T. N. Kumar, Massachusetts General Hospital (United States)
Brian J. Bacskai, Massachusetts General Hospital (United States)
Bradley T. Hyman, Massachusetts General Hospital (United States)

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