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Bioconjug Chem. 2017 May 17;28(5):1356-1362. doi: 10.1021/acs.bioconjchem.7b00047. Epub 2017 Apr 26.

Tethered Fluorogen Assay to Visualize Membrane Apposition in Living Cells.

Author information

1
Department of Biological Sciences, ‡Department of Chemistry, and §Molecular Biosensor and Imaging Center, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.

Abstract

We describe proof-of-concept for a novel approach for visualizing regions of close apposition between the surfaces of living cells. A membrane-anchored protein with high affinity for a chemical ligand is expressed on the surface of one set of cells, and the cells are co-cultured with a second set of cells that express a membrane-anchored fluorogen-activating protein (FAP). The co-cultured cells are incubated with a bivalent reagent composed of fluorogen linked to the high-affinity ligand, with the concentration of the bivalent reagent chosen to be less than the binding constant for the FAP-fluorogen pair but greater than the binding constant for the ligand-high-affinity protein pair. In these conditions, strong FAP signal is observed only in regions of close proximity between membranes of the two classes of cell, where high local concentration of fluorogen favors binding to the FAP.

PMID:
28414915
DOI:
10.1021/acs.bioconjchem.7b00047
[Indexed for MEDLINE]

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