Janeliafluor halotag ligands

For HaloTag- or SnapTag-labeled single-particle imaging, JF646-HaloTag ligand (Janelia Fluor HaloTag Ligands, Promega, GA1120) and TMR-SnapTag ligand (SNAP-Cell TMR-Star, NEB, S9105S) diluted in DMSO were added to the cell culture media and incubated at 37 °C and 5% CO₂. Non-bound ligands were removed after a 15 min incubation with cell culture media, which was repeated twice. For single-particle tracking, we sparsely labeled the protein tags with 1 nM for 7 min for each ligand, which is 100 times more diluted than the usual protocol.
For dual-color single-particle tracking of misfolded polypeptides and YTHDF2, OptoSplitII (Cairn Research) was used to simultaneously image two different wavelengths. To improve the detected signals, we labeled the protein tags with slightly higher concentrations: 20 nM for TMR-SnapTag and 10 nM for JF646-HaloTag.