Tag-stabilized fluorescent protein binders for endogenous cell type-specific protein labelling
- 1Biozentrum, University of Basel
Abstract
Protein labelling using genetically encoded fluorescent protein fusions has revolutionized the study of protein localization and function. However, visualizing endogenous proteins in specific cell types remains challenging, as existing approaches often require overexpression or laborious genetic engineering. To address these limitations, we developed a library of fluorescent protein binders using existing nanobodies and single-chain variable fragments that recognize fluorescent proteins and short peptide tags, allowing for modular, cell type-specific labelling of genetically tagged endogenous proteins. We found that fluorescent protein binders generate background fluorescence in the absence of the target protein, reducing signal specificity. To overcome this limitation, we generated destabilized fluorescent protein binders directed against GFP, ALFA, GCN4 peptide and V5. These binders display increased antigen-dependent stability and reduced background fluorescence, thereby improving the signal-to-noise ratio of endogenous protein visualization. We demonstrate the detection of endogenous proteins in drosophila larvae and embryos, zebrafish embryos, as well as in specific neuronal populations of the adult drosophila brain. In the brain, tissue-specific labelling revealed previously unknown protein expression and subcellular localization in sleep-regulatory neurons. Together, our panel of tag-stabilized fluorescent protein binders provides a versatile, ready-to-use toolkit for background-free, cell type-specific visualization of endogenous proteins.
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BibTeX
@misc{schnider2026tagstabilized,
title = {{Tag-stabilized fluorescent protein binders for endogenous cell type-specific protein labelling}},
author = {S. T. Schnider and C. Kring and L. Maggi and A. M. Vigano and M. Affolter and G. Aguilar and A. Kempf},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.29.754891},
url = {https://www.biorxiv.org/content/10.64898/2026.09.29.754891v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Tag-stabilized fluorescent protein binders for endogenous cell type-specific protein labelling
AU - S. T. Schnider
AU - C. Kring
AU - L. Maggi
AU - A. M. Vigano
AU - M. Affolter
AU - G. Aguilar
AU - A. Kempf
PY - 2026
DA - 2026/09/29
PB - bioRxiv
DO - 10.64898/2026.09.29.754891
UR - https://www.biorxiv.org/content/10.64898/2026.09.29.754891v1
AB - Protein labelling using genetically encoded fluorescent protein fusions has revolutionized the study of protein localization and function. However, visualizing endogenous proteins in specific cell types remains challenging, as existing approaches often require overexpression or laborious genetic engineering. To address these limitations, we developed a library of fluorescent protein binders using existing nanobodies and single-chain variable fragments that recognize fluorescent proteins and short peptide tags, allowing for modular, cell type-specific labelling of genetically tagged endogenous proteins. We found that fluorescent protein binders generate background fluorescence in the absence of the target protein, reducing signal specificity. To overcome this limitation, we generated destabilized fluorescent protein binders directed against GFP, ALFA, GCN4 peptide and V5. These binders display increased antigen-dependent stability and reduced background fluorescence, thereby improving the signal-to-noise ratio of endogenous protein visualization. We demonstrate the detection of endogenous proteins in drosophila larvae and embryos, zebrafish embryos, as well as in specific neuronal populations of the adult drosophila brain. In the brain, tissue-specific labelling revealed previously unknown protein expression and subcellular localization in sleep-regulatory neurons. Together, our panel of tag-stabilized fluorescent protein binders provides a versatile, ready-to-use toolkit for background-free, cell type-specific visualization of endogenous proteins.
ER -