Outstanding Performance for Imaging HaloTag-Labeled Enterococcus PG Hydrolases

July 18, 2026

BlueskyReddit
The Scripps Research Institute
Immunology Microbiology
Postdoctoral Researcher

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Ease-of-Optimization

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Company:

Promega

Product Name:

Janelia Fluor® JFX650 HaloTag® Ligand

Catalog Number:

HT1070

Our laboratory investigates the cell wall biology of Enterococcus faecium, with a particular focus on secreted peptidoglycan hydrolases involved in cell wall remodeling and division. I evaluated the Janelia Fluor® JFX650 HaloTag® Ligand for live-cell imaging of a chromosomally HaloTag-tagged secreted cell wall hydrolase and compared its performance with several other HaloTag fluorophores. Among all the dyes tested, JFX650 consistently provided the brightest, sharpest, and most reliable labeling.

Experimental Design and Results Summary

Application

Live-cell fluorescence microscopy for localization

Starting Material

Live Enterococcus faecium cells expressing a chromosomally encoded HaloTag fusion to a secreted cell wall hydrolase

Tips

Cells were labeled with 1 µM JFX650 HaloTag ligand for 15 minutes at 37°C. The dye readily penetrated the cells and generated a strong fluorescent signal without requiring any protocol optimization.

Results Summary

JFX650 produced the brightest and most well-defined HaloTag-specific signal of all the HaloTag ligands evaluated. The fluorescence closely matched the expected localization of the secreted cell wall hydrolase at the cell envelope and showed excellent agreement with an independent cell wall stain used as a reference. The high signal-to-background ratio and lack of nonspecific labeling enabled confident visualization of the protein in live Enterococcus faecium cells.

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Summary

The Good

High signal-to-background ratio.

The Bad

NA

The Bottom Line

JFX650 was the best-performing HaloTag ligand in our hands for imaging a secreted cell wall hydrolase in Enterococcus faecium. It combined excellent brightness, outstanding labeling specificity, minimal background fluorescence, and accurate localization, making it our preferred choice for live-cell fluorescence microscopy of HaloTag-tagged cell wall proteins.

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