Automated Fluorescence Lifetime Imaging Plate Reader and Its Application to Förster Resonant Energy Transfer Readout of Gag Protein Aggregation

Overview

Journal J Biophotonics

Date 2012 Nov 28

PMID 23184449

Citations 17

Authors

Dominic Alibhai

Douglas J Kelly

Sean Warren

Sunil Kumar

Anca Margineau

Remigiusz A Serwa

Emmanuelle Thinon

Yuriy Alexandrov

Edward J Murray

Frank Stuhmeier

Edward W Tate

Mark A A Neil

Chris Dunsby

Paul M W French

Affiliations

Soon will be listed here.

Abstract

Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z' factors exceeding 0.6 are realised for this FLIM FRET assay.

Citing Articles

High-throughput, multi-parametric, and correlative fluorescence lifetime imaging.

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Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of α-Synuclein and Polyglutamine in Aging .

Laine R, Sinnige T, Ma K, Haack A, Poudel C, Gaida P ACS Chem Biol. 2019; 14(7):1628-1636.

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Combining TIR and FRET in Molecular Test Systems.

Schneckenburger H, Weber P, Wagner M, Enderle S, Kalthof B, Schneider L Int J Mol Sci. 2019; 20(3).

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Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells.

Guo W, Kumar S, Gorlitz F, Garcia E, Alexandrov Y, Munro I SLAS Technol. 2019; 24(3):308-320.

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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

Gorlitz F, Kelly D, Warren S, Alibhai D, West L, Kumar S J Vis Exp. 2017; (119).

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