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| Item Type: | Article |
|---|---|
| Title: | Penta-ALFA-tagged substrates for self-labelling tags allow signal enhancement in microscopy |
| Creators Name: | Ghosh, S., Birke, R., Natarajan, A.K. and Broichhagen, J. |
| Abstract: | Self-labelling proteins like SNAP- and HaloTag have advanced imaging in life sciences by enabling live-cell labeling with fluorophore-conjugated substrates. However, the typical one-fluorophore-per-protein system limits signal intensity. To address this, we developed a strategy using the ALFA-tag system, a 13-amino acid peptide recognized by a bio-orthogonal and fluorescently labelled nanobody, for signal amplification. We synthesized a pentavalent ALFA(5) peptide and used an azidolysine for conjugation with a Cy5-modified SNAP- or HaloTag ligand through strain-promoted click chemistry. In vitro measurements on SDS-PAGE showed labelling, and the peptides covalently reacted with their respective tag. HEK293 cells expressing SNAP- and HaloTag-mGluR2 fusion proteins were labeled with ALFA(5)-Cy5 substrates, and confocal microscopy revealed a significant enhancement in the far-red signal intensity upon nanobody addition, as quantified by integrated signal density ratios. Comparisons between SNAP- and HaloTag substrates showed superior performance for the latter, achieving better signal-to-noise and signal-to-background ratios, as well as overall signal intensity in plasma membrane-localized regions. Our results demonstrate the potential of ALFA-tag-based systems to amplify SLP fluorescent signals. This strategy combines the photostability of synthetic fluorophores with multivalent labeling, providing a powerful tool for advanced imaging applications including super-resolution in cells. Its versatility is expandable across diverse protein systems and colors. |
| Keywords: | ALFA-Tag, Cell Imaging, Confocal Microscopy, Fluorescent Labeling, Nanobody, Self-Labelling Proteins (SLPs), Signal Amplification |
| Source: | Journal of Peptide Science |
| ISSN: | 1075-2617 |
| Publisher: | Wiley |
| Volume: | 31 |
| Number: | 5 |
| Page Range: | e70015 |
| Date: | May 2025 |
| Official Publication: | https://doi.org/10.1002/psc.70015 |
| PubMed: | View item in PubMed |
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