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Bioconjug Chem. 2019 May 15;30(5):1309-1313. doi: 10.1021/acs.bioconjchem.9b00203. Epub 2019 Apr 17.

Fluorogenic Photoaffinity Labeling of Proteins in Living Cells.

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1
Department of Chemistry , Emory University , Atlanta , Georgia 30322 , United States.

Abstract

Genetically encoded fluorescent proteins or small-molecule probes that recognize specific protein binding partners can be used to label proteins to study their localization and function with fluorescence microscopy. However, these approaches are limited in signal-to-background resolution and the ability to temporally control labeling. Herein, we describe a covalent protein labeling technique using a fluorogenic malachite green probe functionalized with a photoreactive cross-linker. This enables a controlled covalent attachment to a genetically encodable fluorogen activating protein (FAP) with low background signal. We demonstrate covalent labeling of a protein in vitro as well as in live mammalian cells. This method is straightforward, displays high labeling specificity, and results in improved signal-to-background ratios in photoaffinity labeling of target proteins. Additionally, this probe provides temporal control over reactivity, enabling future applications in real-time monitoring of cellular events.

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