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

• DOI: 10.1007/978-1-4939-7265-4_15
• Publication Date: 2017-09-18T13:04:07Z
• Authors: Jonathan B. Grimm; Timothy A. Brown; Brian P. English; Timothée Lionnet; Luke D. Lavis
• Keywords: Alexa Fluor; Snap

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.