Penta-ALFA-Tagged Substrates for Self-Labelling Tags Allow Signal Enhancement in Microscopy.

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 ALFA₅ 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

SUBMITTER: Ghosh S 

PROVIDER: S-EPMC11994250 | biostudies-literature | 2025 May

REPOSITORIES: biostudies-literature