Penta‐ALFA‐Tagged Substrates for Self‐Labelling Tags Allow Signal Enhancement in Microscopy DOI Creative Commons
Souvik Ghosh,

Ramona Birke,

Ashwin Karthick Natarajan

и другие.

Journal of Peptide Science, Год журнала: 2025, Номер 31(5)

Опубликована: Апрель 13, 2025

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 ALFA-tag system, 13-amino acid peptide recognized bio-orthogonal fluorescently labelled nanobody, for amplification. We synthesized pentavalent ALFA5 used an azidolysine conjugation Cy5-modified or ligand through strain-promoted click chemistry. In vitro measurements on SDS-PAGE showed labelling, peptides covalently reacted their respective tag. HEK293 cells expressing HaloTag-mGluR2 fusion were labeled ALFA5-Cy5 substrates, confocal microscopy revealed significant enhancement far-red intensity upon nanobody addition, as quantified integrated density ratios. Comparisons between substrates superior performance latter, achieving better signal-to-noise signal-to-background ratios, well overall plasma membrane-localized regions. Our results demonstrate potential of ALFA-tag-based systems to amplify SLP fluorescent signals. This combines photostability synthetic fluorophores multivalent labeling, providing powerful tool applications including super-resolution cells. Its versatility is expandable across diverse protein colors.

Язык: Английский

Penta‐ALFA‐Tagged Substrates for Self‐Labelling Tags Allow Signal Enhancement in Microscopy DOI Creative Commons
Souvik Ghosh,

Ramona Birke,

Ashwin Karthick Natarajan

и другие.

Journal of Peptide Science, Год журнала: 2025, Номер 31(5)

Опубликована: Апрель 13, 2025

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 ALFA-tag system, 13-amino acid peptide recognized bio-orthogonal fluorescently labelled nanobody, for amplification. We synthesized pentavalent ALFA5 used an azidolysine conjugation Cy5-modified or ligand through strain-promoted click chemistry. In vitro measurements on SDS-PAGE showed labelling, peptides covalently reacted their respective tag. HEK293 cells expressing HaloTag-mGluR2 fusion were labeled ALFA5-Cy5 substrates, confocal microscopy revealed significant enhancement far-red intensity upon nanobody addition, as quantified integrated density ratios. Comparisons between substrates superior performance latter, achieving better signal-to-noise signal-to-background ratios, well overall plasma membrane-localized regions. Our results demonstrate potential of ALFA-tag-based systems to amplify SLP fluorescent signals. This combines photostability synthetic fluorophores multivalent labeling, providing powerful tool applications including super-resolution cells. Its versatility is expandable across diverse protein colors.

Язык: Английский

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