PT  - JOURNAL ARTICLE
AU  - Falk Schneider
AU  - Christian Eggeling
AU  - Erdinc Sezgin
TI  - Influence of nanobody binding on fluorescence emission, mobility and organization of GFP-tagged proteins
AID  - 10.1101/2020.06.11.146274
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.06.11.146274
4099  - http://biorxiv.org/content/early/2020/06/11/2020.06.11.146274.short
4100  - http://biorxiv.org/content/early/2020/06/11/2020.06.11.146274.full
AB  - Advanced fluorescence microscopy studies require specific and monovalent molecular labelling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labelled nanobodies against commonly employed fluorescent proteins. However, very little is known how these nanobodies influence their target molecules. Here, we observed clear changes of the fluorescence properties, mobility and organisation of green fluorescent protein (GFP) tagged proteins after labelling with an anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently-labelled nanobodies with the GFP-labelled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, oligomerized form of the fluorescent proteins, promoting disassembly into more monomeric forms after binding. Our findings show that great care must be taken when using nanobodies for studying dynamic and quantitative protein organisation.Competing Interest StatementThe authors have declared no competing interest.