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 GFP.

In 2022, we reported a method based on use of small epitopes (127D01 and VHH05) that are recognized by specific nanobodies (Nb127D01 and NbVHH05). This ‘NanoTag’ system allows you to first tag an endogenous gene with the 127D01 or VHH05 epitope, then use purified or in vivo expressed versions of Nb127D01 or NbVHH05 to detect the tagged protein.

The Xu and Kim et al. 2022 eLife research publication is available as a PDF download attached to this news post (see below). We subsequently published a protocol manuscript: Kim and Xu et al. 2022 in Current Protocols. Both publications can be accessed at the Perrimon lab publications page.

One of the most common inquiries we have received since publication of the paper is requests for purified, fluorophore-conjugated Nb127D01 and NbVHH05 nanobodies. For the study reported in eLife, we made a tiny amount of each fluorophore-conjugated nanobody. Thus, we do not have material available to share.

However, these can be generated in your lab using commercially available antibody labeling kits for conjugating fluorophores to nanobodies purified from bacteria: the Mix-n-Stain CF 555 Antibody Labeling Kit or the Mix-n-Stain CF 647 Antibody Labeling Kit.

Because 50 ml of bacterial culture usually gives ~1 mg of purified protein, the amount needed for fluorophore conjugation can be easily prepared. Please check the protocol for periplasmic protein expression and purification in our protocol publication in Current Protocols. If you have further questions about preparing reagents, please contact Ah-Ram Kim, PhD. His email address is available on the Perrimon lab 'People' page.

If the NanoTag system works well in your system and is used in a future publication, please cite the research and/or protocol publication we've mentioned. We also ask that you acknowledge all relevant repositories from which you received materials and our technology group, the DRSC-BTRR at Harvard Medical School.

Note added 2023-04: A preprint from the Gelfand lab includes knock-in of 3xVHH05 and visualization in vivo in Drosophila using NbVHH05-EGFP (see Fig. 4). Exciting to see our DRSC-BTRR tech in use 'in the wild'!
Lu, Lakonishok and Gelfand (2023) BioRxiv: "Drosophila oocyte specification is maintained by the dynamic duo of microtubule polymerase Mini spindles/XMAP215 and dynein" https://www.biorxiv.org/content/10.1101/2023.03.09.531953v1