Issue 7, 2015

Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins

Abstract

Agonist-promoted G-protein coupled receptor (GPCR) endocytosis and recycling plays an important role in many signaling events in the cell. However, the approaches that allow fast and quantitative analysis of such processes still remain limited. Here we report an improved labeling approach based on the genetic fusion of a fluorogen activating protein (FAP) to a GPCR and binding of a sulfonated analog of the malachite green (MG) fluorogen to rapidly and selectively label cell surface receptors. Fluorescence microscopy and flow cytometry demonstrate that this dye does not cross the plasma membrane, binds with high affinity to a dL5** FAP-GPCR fusion construct, activating tagged surface receptors within seconds of addition. The ability to rapidly and selectively label cell surface receptors with a fluorogenic genetically encoded tag allows quantitative imaging and analysis of highly dynamic processes like receptor endocytosis and recycling.

Graphical abstract: Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins

Supplementary files

Article information

Article type
Paper
Submitted
30 Oct 2014
Accepted
09 Dec 2014
First published
10 Dec 2014

Org. Biomol. Chem., 2015,13, 2078-2086

Author version available

Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins

Q. Yan, B. F. Schmidt, L. A. Perkins, M. Naganbabu, S. Saurabh, S. K. Andreko and M. P. Bruchez, Org. Biomol. Chem., 2015, 13, 2078 DOI: 10.1039/C4OB02309A

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