HaloTag labeling

HaloTag Labeling in Yeast

This protocol describes how to label a HaloTag fusion protein in yeast cells for confocal microscopy.

Labeling is best performed in drug-sensitive pdr1∆ pdr3∆ cells, although it might work under some conditions in wild-type cells.

Treatment of coverglass-bottom dish (MatTek P35G-0.170-14-C) with Concanavalin A (ConA)

Thaw an aliquot of 2 mg/ml ConA (in H₂O, Sigma C-2010, kept at -80°C in 500 μl aliquots). Vortex the solution, and centrifuge at 15,000xg for 5 min to precipitate any undissolved ConA. Pipette 250 μl of ConA solution onto the dish coverslip, let it stand for 15 min, remove the ConA, wash with 1 ml pure H₂O, and allow the dish to dry well before attempting to attach cells. Same day ConA-treated dishes work best.

HaloTag labeling

Stock of the dye: 1 mM JFX₆₄₆- or JFX₆₅₀-HaloTag ligand (Janelia dyes, obtained from Luke Lavis) in anhydrous DMSO (Invitrogen D12345), stored in 1 μl aliquots at -80°C protected from light.

1. Grow an overnight 5 ml yeast culture to log phase in microscopy medium (we use our custom Nonfluorescent Synthetic Dextrose medium = NSD, but regular SD should also work) in a 50 ml baffled flask at 23°C, shaking at 200 rpm. Aim for an OD of 0.5-0.6.
2. When ready to start labeling, add 1 μl of the JFX-HaloTag ligand to 300 μl fresh microscopy medium and mix well by pipetting and vortexing. Remove any precipitate by centrifugation at 15,000xg for 5 min.
3. Add 300 μl of the centrifuged ligand-containing medium to 700 μl of yeast culture in a 15 ml culture tube, yielding a final dye concentra;on of 1 µM^*1. Mix by vortexing briefly. Incubate for 45 min to 1 h^*2 at 23°C, shaking at 200 rpm and protected from light.
4. Remove the excess dye by filtration through a 0.22 µm filter (Millipore Sigma, Millex-GV, SLGV013SL) using a syringe (BD, 3 ml Luer-Lok syringe, 309657). Wash the cells that remain on the filter 1-2 times^*3 by passing 3 ml of fresh culture medium through the filter.
5. Resuspend the cells from the filter by carefully pipetting 125 µl of medium up and down on the filter several times. Repeat with a second aliquot of 125 µl microscopy medium and combine the two aliquots of resuspended cells.
6. Place the cell suspension onto a ConA-coated coverglass-bottom dish. Wait 5–10 min. Remove the cells that were not attached, wash gently with 1 ml microscopy medium, and add 2.0–2.5 ml microscopy medium to the dish. Then proceed with the microscopy^{*4, 5}.

^*1 Using double the concentration (2 μM final) may improve labeling.

^*2 30 min can work but longer incubations may improve labeling.

^*3 If necessary, washing can be avoided without major compromise in the signal-to-background ratio. This effect might be due to the equilibrium between the colorless, nonfluorescent, “closed” lactone form and the colored, fluorescent, “open” zwitterionic form of the dye in aqueous solution. For example, JF_646-HaloTag ligand shows a 21-fold increase in absorbance when bound to the HaloTag protein (see Grimm et al, Nature Methods, 2017; DOI: 10.1038/NMETH.4403).

^*4 For optimal imaging, observe the cells immediately after labeling and for up to an hour after washing out the dye. Prolonged incubation without the dye may result in a decline of labeled HaloTag due to protein turnover.

^*5 JFX₆₄₆/JFX₆₅₀-HaloTag-labeled fusion proteins can be observed by fluorescence microscopy using a 640 nm excitation laser and a 650–750 nm collection window. We use a HyDX detector with a Stellaris Falcon 8 confocal, using photon counting mode and a 0.6–8 gating.