Automated nanoscale flow cytometry for assessing protein-protein interactions

2016 ◽  
Vol 89 (9) ◽  
pp. 835-843 ◽  
Author(s):  
Kerstin von Kolontaj ◽  
Gabor L. Horvath ◽  
Eicke Latz ◽  
Martin Büscher
Keyword(s):  
Flow Cytometry ◽  
Protein Interactions ◽  
Protein Protein Interactions

scholarly journals A Flow Cytometry-Based FRET Assay to Identify and Analyse Protein-Protein Interactions in Living Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (2) ◽  
pp. e9344 ◽  
Author(s):  
Carina Banning ◽  
Jörg Votteler ◽  
Dirk Hoffmann ◽  
Herwig Koppensteiner ◽  
Martin Warmer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Interactions ◽  
Living Cells ◽  
Protein Protein Interactions

scholarly journals Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

Theranostics ◽  
2019 ◽  
Vol 9 (19) ◽  
pp. 5444-5463 ◽  
Author(s):  
Verena Trümper ◽  
Andreas von Knethen ◽  
Annegret Preuß ◽  
Eugeny Ermilov ◽  
Steffen Hackbarth ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Interactions ◽  
Living Cells ◽  
Protein Protein Interactions

scholarly journals A quantitative tri-fluorescent yeast two-hybrid system: from flow cytometry to in-cellula affinities

2019 ◽  
Author(s):  
David Cluet ◽  
Ikram Amri ◽  
Blandine Vergier ◽  
Jérémie Léault ◽  
Clémence Grosjean ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Hybrid System ◽  
Protein Interactions ◽  
Technological Advancement ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
High Throughput Analysis ◽  
Expression Levels ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

AbstractWe present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on PPIs with different affinities. After only two hours of reaction, expression of the reporter can easily be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflects the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated high-throughput analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

scholarly journals Titration of in-cellula affinities of protein-protein interactions

2020 ◽  
Author(s):  
David Cluet ◽  
Blandine Vergier ◽  
Nicolas-Pierre Levy ◽  
Lucie Dehau ◽  
Alexandre Thurman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Interactions ◽  
Dissociation Constants ◽  
Single Cell Level ◽  
Interacting Proteins ◽  
Protein Protein Interactions ◽  
Cell Level ◽  
Simultaneous Quantification ◽  
Genetic Assay

A genetic assay permits simultaneous quantification of two interacting proteins and their bound fraction at the single-cell level using flow cytometry. In-cellula affinities of protein-protein interactions can be extracted from the acquired data through a titration-like analysis. The applicability of this approach is demonstrated on a diverse set of interactions with proteins from different families and organisms and with in-vitro dissociation constants ranging from picomolar to micromolar.

Flow Cytometry for Biotechnology

2005 ◽  
Keyword(s):  
Flow Cytometry ◽  
Drug Discovery ◽  
Laser Beam ◽  
Protein Interactions ◽  
Fluorescent Dyes ◽  
Vaccine Development ◽  
Genomic Sequence ◽  
Sequence Information ◽  
Protein Protein Interactions ◽  
Pharmacology And Toxicology

Flow cytometry is a sensitive and quantitative platform for the measurement of particle fluorescence. In flow cytometry, the particles in a sample flow in single file through a focused laser beam at rates of hundreds to thousands of particles per second. During the time each particle is in the laser beam, on the order of ten microseconds, one or more fluorescent dyes associated with that particle are excited. The fluorescence emitted from each particle is collected through a microscope objective, spectrally filtered, and detected with photomultiplier tubes. Flow cytometry is uniquely capable of the precise and quantitative molecular analysis of genomic sequence information, interactions between purified biomolecules and cellular function. Combined with automated sample handling for increased sample throughput, these features make flow cytometry a versatile platform with applications at many stages of drug discovery. Traditionally, the particles studied are cells, especially blood cells; flow cytometry is used extensively in immunology. This volume shows how flow cytometry is integrated into modern biotechnology, dealing with issues of throughput, content, sensitivity, and high throughput informatics with applications in genomics, proteomics and protein-protein interactions, drug discovery, vaccine development, plant and reproductive biology, pharmacology and toxicology, cell-cell interactions and protein engineering.

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