Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments

  • Jonathan B. Grimm
  • Timothy A. Brown
  • Brian P. English
  • Timothée Lionnet
  • Luke D. LavisEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1663)


The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the “Janelia Fluor” dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.

Key words

Fluorophore HaloTag SNAP-tag Fluorescence microscopy Organic chemistry Single-molecule imaging 


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jonathan B. Grimm
    • 1
  • Timothy A. Brown
    • 1
  • Brian P. English
    • 1
  • Timothée Lionnet
    • 1
  • Luke D. Lavis
    • 1
    Email author
  1. 1.Janelia Research CampusHoward Hughes Medical InstituteAshburnUSA

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