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© 2020 The Author(s) Advanced fluorescence microscopy studies require specific and monovalent molecular labeling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labeled nanobodies against commonly employed fluorescent proteins (FPs). However, very little is known how these nanobodies influence their target molecules. Here, we tested commercially available nanobodies and observed clear changes of the fluorescence properties, mobility and organization of green fluorescent protein (GFP) tagged proteins after labeling with the anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently labeled nanobodies with the GFP-labeled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, likely oligomerized, form of the FPs, promoting disassembly into monomeric form after binding. Our findings have significant implications on the application of nanobodies and GFP labeling for studying dynamic and quantitative protein organization in the plasma membrane of living cells using advanced imaging techniques.

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