Protein binders

Figure 1 from Xu, Kim et al 2022 in eLife

Light up your flies! Reagent and protocol information for application of our NanoTag system for epitope tagging in Drosophila

March 23, 2023

One of the areas of interest for our technology development group is nanobodies. These small, single-chain antibodies are particularly attractive for Drosophila research as in addition to being used for standard immune-technologies such as immunoblots and immunostaining of tissues, they can also be expressed in vivo as fusions to fluorescent proteins (‘chromobodies’) or functional domains (e.g., for degradation or re-localization).

Past applications of this technology in Drosophila relied on availability of nanobodies targeting a specific protein or use of nanobodies targeting...

Read more about Light up your flies! Reagent and protocol information for application of our NanoTag system for epitope tagging in Drosophila
Jun Xu, Ah-Ram Kim, Ross W Cheloha, Fabian A Fischer, Joshua Shing Shun Li, Yuan Feng, Emily Stoneburner, Richard Binari, Stephanie E Mohr, Jonathan Zirin, Hidde L Ploegh, and Norbert Perrimon. 2022. “Protein visualization and manipulation in through the use of epitope tags recognized by nanobodies.” Elife, 11.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.
Jun Xu, Ah-Ram Kim, Ross W. Cheloha, Fabian A. Fischer, Joshua Shing Shun Li, Yuan Feng, Emily Stoneburner, Richard Binari, Stephanie E. Mohr, Jonathan Zirin, Hidde Ploegh, and Norbert Perrimon. 9/29/2021. “Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies.” bioRxiv.Abstract
Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags installed on 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 combine the advantages of short epitopes (NanoTags) and nanobodies specific for them by characterizing two short (<15 aa) tags, 127D01 and VHH05, which are high-affinity targets of nanobodies. We demonstrate that these NanoTags and the nanobodies that recognize them can be used 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.Competing Interest StatementThe authors have declared no competing interest.