1 September 2006 Time-domain fluorescent plate reader for cell based protein-protein interaction and protein conformation assays
Author Affiliations +
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.
© (2006) Society of Photo-Optical Instrumentation Engineers (SPIE)
Phill B. Jones, Phill B. Jones, Lauren Herl, Lauren Herl, Oksana Berezovska, Oksana Berezovska, Anand T. N. Kumar, Anand T. N. Kumar, Brian J. Bacskai, Brian J. Bacskai, Bradley T. Hyman, Bradley T. Hyman, } "Time-domain fluorescent plate reader for cell based protein-protein interaction and protein conformation assays," Journal of Biomedical Optics 11(5), 054024 (1 September 2006). https://doi.org/10.1117/1.2363367 . Submission:
JOURNAL ARTICLE
10 PAGES


SHARE
Back to Top