HaloTag-Mediated Protein Labeling and Imaging

HaloTag labeling was performed as previously described (Casler et al., 2019 (link)). To visualize proteins fused to HaloTag, JFX₆₄₆ or JFX₆₅₀ ligand (Grimm et al., 2021 (link)), kindly provided by Luke Lavis (Janelia Research Campus, Ashburn, VA), was diluted 1:1,000 from a 1-mM stock in DMSO in 0.5 ml of culture medium to give a final concentration of 1 µM. The medium was cleared of any precipitate by spinning at 17,000×g (13,000 rpm) in a microcentrifuge for 1 min. Then the cleared medium containing ligand was added to 0.5 ml of log-phase yeast culture, and the cells were incubated with shaking at 23°C for 30 min. Excess dye was removed by filtration through and washing on a 0.22-µm syringe filter (Millipore; catalog #SLGV004SL). The washed cells were resuspended in NSD and attached to a concanavalin A–coated coverglass-bottom dish. Videos were captured immediately by confocal microscopy.