Fluorescent dye JF-646 (Grimm et al., 2015 (link)) NHS-ester building block (TOCRIS) was conjugated with Halo-tag ligand amine O4 (Promega) by synthetic chemistry according to published protocols (Grimm et al., 2017 (link)). Briefly, 1.5 equivalents of amine O4 ligand were added to one equivalent of the JF-646 NHS-ester in DMF followed by adding 5% triethylamine. The reaction was vigorously stirred for 16 hr at room temperature and the product was purified by silica gel chromatography, dried by SpeedVac (ThermoFisher), and reconstituted in DMSO.
For optical trapping/confocal microscopy assays, HaloTag Alexa Fluor 488 Ligand (Promega) was utilized as described above, followed by desalting through a PD SpinTrap G-25 column (GE Healthcare) according to the manufacturer’s protocol to remove unreacted dye before use. To label the Halo-tagged actin-binding proteins with Halo-JF-646 for TIRF microscopy assays, two equivalents of synthesized Halo-JF-646 dye was added to the protein solution, followed by incubation for at least 2 hr in the dark at 4°C before use. Subsequent removal of excess dye was not required, as JF-646 is a fluorogenic dye (Grimm et al., 2015 (link)).
For optical trapping/confocal microscopy assays, HaloTag Alexa Fluor 488 Ligand (Promega) was utilized as described above, followed by desalting through a PD SpinTrap G-25 column (GE Healthcare) according to the manufacturer’s protocol to remove unreacted dye before use. To label the Halo-tagged actin-binding proteins with Halo-JF-646 for TIRF microscopy assays, two equivalents of synthesized Halo-JF-646 dye was added to the protein solution, followed by incubation for at least 2 hr in the dark at 4°C before use. Subsequent removal of excess dye was not required, as JF-646 is a fluorogenic dye (Grimm et al., 2015 (link)).