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

Methods Mol Biol. 2017:1663:179-188. doi: 10.1007/978-1-4939-7265-4_15.

Abstract

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.

Keywords: Fluorescence microscopy; Fluorophore; HaloTag; Organic chemistry; SNAP-tag; Single-molecule imaging.

MeSH terms

  • Animals
  • Fluorescent Dyes / chemical synthesis*
  • Fluorescent Dyes / chemistry
  • Humans
  • Ligands
  • Mammals
  • Microscopy, Fluorescence / methods*
  • Molecular Structure
  • Organic Chemistry Phenomena
  • Single Molecule Imaging
  • Staining and Labeling

Substances

  • Fluorescent Dyes
  • Ligands