Halo ligand

The Munc13-1-Halo proteins were labeled by incubating the proteins with Alexa488 or Alexa660 conjugated with a Halo ligand from Promega, as described before (12 (link)). The protein was first centrifuged at 14,000 rpm for 20 min at 4 °C to remove any precipitation. Fluorescence dye was added into the protein solution at dye:protein = 5:1 molar ratio and the mixture was incubated for 30 min at room temperature with gentle rotation. Unreacted dye was removed by passing through the PD MidiTrap G-25 column (GE Healthcare) three times at room temperature. The labeling efficiencies were about 97%.

Halo fusion proteins were visualized in cells by staining with fluorescent Halo ligand (Promega GA1110). Cells were seeded on polyD lysine-coated coverslips (neuVitro) in full growth media supplemented with 2 μg/mL doxycycline to induce Halo transgene expression for 48 hr. Following a PBS wash, cells were incubated with Halo ligand Janelia Fluor 549 (Promega GA1110) at 25 nM for 30 min at room temperature. Following PBS wash, cells were fixed with 3.7% formaldehyde for 20 min at room temperature. Cell nuclei were stained with 100 ng/mL DAPI (Sigma-Aldrich D9542) for 15 min, and then cells were washed again with PBS. Coverslips were mounted with fluoromount G (SouthernBiotech 0100–01) and imaged at ×60 magnification with a Deltavision Elite widefield fluorescence microscope (GE). DAPI was imaged with 5% transmittance with an exposure of 0.3 s. Halo staining was visualized in the TRITC channel with 10% transmittance and an exposure of 0.2 s. Z stacks were collected of approximately 68 images, 0.2 µm apart for a total thickness of 13.6 µm.