ArticleBiophysical journal2015
A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.
Article in Biophysical journal, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Multi-parameter fluorescence lifetime imaging for high-noise neuroscience applications.Biomedical optics express · 2026Article
- Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies.The Journal of biological chemistry · 2020Article
- Analysis of Brightness of a Single Fluorophore for Quantitative Characterization of Biochemical Reactions.The journal of physical chemistry. B · 2020Article
- Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of α-Synuclein and Polyglutamine in AgingACS chemical biology · 2019Article
- Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells.Scientific reports · 2019Article
- Determination of oligomerization state of Drp1 protein in living cells at nanomolar concentrations.Scientific reports · 2019Article
- Phasor analysis of NADH FLIM identifies pharmacological disruptions to mitochondrial metabolic processes in the rodent cerebral cortex.PloS one · 2018Article
- Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy.The journal of physical chemistry. C, Nanomaterials and interfaces · 2017Article
- Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.Analytical chemistry · 2017Article
- Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in Vitro and in Cells.Nano letters · 2017Article
- Article
- Translation Elongation Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin.Scientific reports · 2016Article
- Current Approaches Toward Quantitative Mapping of the Interactome.Frontiers in genetics · 2016Article
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Authors and funding
9 authors.
Funding
Abstract
FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.
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