Halo-tag Labeling

Cell expressing the Halo protein or a Halo-tagged fusion protein can be labeled live, without fixation using Halo ligands ordered from Promega. This protocol for labeling of live cells with Halo ligand is also found on the Promega website in the “Focus on Imaging Technical Manual.”

Halo-tag cell permeable dyes available in lab

Dye

Attune Channel (excitation/emission max)

Stock Concentration

How to Make

Janelia Fluor 549

YL1 (549/571)

200 µM (1000x)

Dissolve 1 vial of JF549 in 37.8 µL of DMSO. Mix thoroughly by pipetting. Make 1 µL aliquots in 1.7 mL centrifuge tubes and freeze in -20°C.

Janelia Fluor 646

RL1 (646/664)

200 µM (1000x)

Dissolve 1 vial of JF646 in 35.5 µL of DMSO. Mix thoroughly by pipetting. Make 1 µL aliquots in 1.7 mL centrifuge tubes and freeze in -20°C.

Note

Halo ligand is aliquoted and frozen to avoid multiple freeze-thaw cycles, as recommended by Promega.

Note

The Promega documentation recommends diluting the stock solutions to 200x at time of use (a working stock) and replacing one fifth of the media on the cells at time of labeling. AMB has skipped this step, diluted straight to 1000x, and added to cells and it works well.

Other materials needed:

  • Appropriate culture medium

  • Adherent cells to stain

Halo ligand staining

  1. Working in a BSC hood, dilute resuspended Halo ligand in appropriate culture medium 1:1000 to making the Halo-working solution.

  2. Aspirate media from adherent cells and replace media with the Halo-working solution.

    • For the Halo staining a “half” well volume may be used (e.g. 1 mL per well of 6-well plate, 0.5 mL in a 12-well, etc.)

  3. Incubate the cells in Halo-working solution in the incubator (37°C) for 15 minutes to overnight.

Note

AMB has tested 15 minutes, 30 minutes, and 1 hour. All have worked well with no noticeable loss-of-signal in shorter incubation times.

  1. Remove Halo-working solution and proceed to downstream application (imaging, flow, etc.). No wash required.