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 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.
Jones
Phill B
PB
Athinoula A. Martinos Center for Biomedical Imaging, Harvard Medical School, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, USA.
Herl
Lauren
L
Berezovska
Oksana
O
Kumar
Anand T N
AT
Bacskai
Brian J
BJ
Hyman
Bradley T
BT
D000465:Algorithms; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011506:Proteins