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Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies

By Jun Xu, Ah-Ram Kim, Ross W Cheloha, Fabian A Fischer, Joshua Shing Shun Li, Yuan Feng, Emily Stoneburner, Richard Binari, Stephanie Mohr, Jonathan Zirin, Hidde Ploegh, Norbert Perrimon

Posted 17 Apr 2021
bioRxiv DOI: 10.1101/2021.04.16.440240

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15 aa) NanoTag epitopes, 127D01 and VHH05) and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.

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