Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies
- Harvard Medical School, United States
- Boston Children's Hospital, United States
Abstract
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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All data generated or analysed during this study are included in the manuscript and supporting file.
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Funding
National Institute of General Medical Sciences (GM132087)
- Norbert Perrimon
National Research Foundation of Korea (2021R1A6A3A14039622)
- Ah-Ram Kim
Croucher Foundation
- Joshua Shing Shun Li
Howard Hughes Medical Institute
- Norbert Perrimon
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Xu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies
eLife 11:e74326.
https://doi.org/10.7554/eLife.74326
