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Updated: May 13, 2025

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Published on: June 30, 2018

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

Souvik Ghosh¹, Ramona Birke¹, Ashwin Karthick Natarajan²

¹Leibniz-Forschungsinstitut Für Molekulare Pharmakologie (FMP), Berlin, Germany.

Journal of Peptide Science : an Official Publication of the European Peptide Society

|April 13, 2025

Summary

This summary is machine-generated.

Signal amplification for protein imaging was achieved using the ALFA-tag system with a pentavalent peptide and nanobody. This method enhances fluorescent signal intensity in live-cell imaging applications.

Keywords:

ALFA‐tag cell imaging confocal microscopy fluorescent labeling nanobody self‐labelling proteins (SLPs)signal amplification

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Published on: September 24, 2015

Area of Science:

Life Sciences
Biotechnology
Molecular Imaging

Background:

Self-labelling proteins (SLPs) like SNAP-tag and HaloTag enable live-cell imaging but are limited by one-fluorophore-per-protein systems.
Low signal intensity in current SLP methods restricts advanced imaging applications and quantitative analysis.

Purpose of the Study:

To develop a novel signal amplification strategy for SLPs using the ALFA-tag system.
To enhance fluorescent signal intensity in live-cell imaging by overcoming the limitations of single-fluorophore labeling.

Main Methods:

Synthesized a pentavalent ALFA5 peptide for multivalent labeling.
Utilized strain-promoted click chemistry to conjugate azidolysine-modified peptides with SNAP- or HaloTag ligands.
Applied the ALFA5-Cy5 substrate to HEK293 cells expressing SNAP- and HaloTag-mGluR2 fusion proteins for confocal microscopy.

Main Results:

Demonstrated successful labeling and covalent reaction of ALFA5-Cy5 substrates with SNAP- and HaloTag fusion proteins in vitro and in cells.
Observed significant enhancement in far-red fluorescent signal intensity upon nanobody addition, confirmed by confocal microscopy.
HaloTag substrates exhibited superior performance, yielding better signal-to-noise and signal-to-background ratios compared to SNAP-tag.

Conclusions:

The ALFA-tag system provides a powerful strategy for amplifying fluorescent signals in self-labelling protein applications.
This multivalent labeling approach enhances signal intensity, offering a valuable tool for advanced live-cell imaging, including super-resolution microscopy.
The system's versatility allows for expansion across various protein systems and fluorophore colors, broadening its applicability in biological research.