Rapid Labeling Protocol for 1nmol Janelia Fluor® andJanelia Fluor® JFX HaloTag® Ligands
Instructions for Use of Products HT1010, HT1020, HT1030, HT1040, HT1050, HT1060, HT1070, HT1100 and HT1110.

Quick Protocol

This protocol is intended for reconstituting aqueous-buffer-soluble 1nmol Janelia Fluor® and Janelia Fluor® JFX HaloTag®
Ligands and labeling live cells.

Materials to Be Supplied by the User•

optical bottom chamber with cells expressing HaloTag® fusion protein•

complete culture medium appropriate for your cells, at 37°C

•confocal microscope or wide-field fluorescent microscope equipped with appropriate filter sets

•37°C + CO2 cell culture incubator•optional: culture medium, lacking phenol red at 37°C

Protocol1. Equilibrate a vial of 1nmol Janelia Fluor® or Janelia Fluor® JFX HaloTag® Ligand to room temperature.
2. Add 1ml of cell medium or chosen aqueous buffer.
3. Incubate HaloTag® Ligand in medium for 2–3 minutes with intermittent agitation to dissolve the ligand. Do not pipet orvortex to resuspend the ligand. This yields a 5X (1µM) working stock solution.
4. Add the 5X working stock solution to cells at a 1X final concentration of 200nM as a recommended starting point.
Further optimizing of ligand concentration may be necessary (1).
5. Incubate the cells with the Janelia Fluor® or Janelia Fluor® JFX HaloTag® Ligand for 30 minutes at 37°C + CO2 in a cell
culture incubator (2,3).
If using the 1nmol Janelia Fluor® 503 HaloTag® Ligand, incubate with cells for 1 hour at 37°C + CO2 in a cell culture
incubator (2).
6. Aspirate medium and replace with fresh cell medium. Alternatively, replace with phenol red-free medium to minimize
background signal.
7. Transfer to a microscope and capture images.
Table 1. Excitation and Emission Maxima for Janelia Fluor® and Janelia Fluor® JFX HaloTag® Ligands.
Excitation
Maximum

Emission
Maximum

Janelia Fluor® 503 HaloTag® Ligand

503

529

Janelia Fluor 549 HaloTag Ligand

549

571

Janelia Fluor® JFX554 HaloTag® Ligand

554

576

Janelia Fluor 585 HaloTag Ligand

585

609

Janelia Fluor® 635 HaloTag® Ligand

635

652

Janelia Fluor 646 HaloTag Ligand

646

664

Janelia Fluor® JFX650 HaloTag® Ligand650

667

Ligand®

®

®

®

®

®

Notes:a. Lower-expressing cells may require lower ligand concentrations that can enhance the signal-to-background ratio.
b. If using lower ligand concentrations, longer incubation times may be required to reach maximum intensity.
Additional protocol information is available in Technical Manual #TM260, online at: www.promega.com

PAGE 1 • PART# FB248

Rapid Labeling Protocol for 1nmol Janelia Fluor® andJanelia Fluor® JFX HaloTag® Ligands

Quick Protocol

Instructions for Use of Products HT1010, HT1020, HT1030, HT1040, HT1050, HT1060, HT1070, HT1100 and HT1110.

References
1. Grimm, J.B., et al. (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat.
Methods 12, 244–50.
2. Grimm, J.B., et al. (2017) A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat Methods 14,987–94.
3. Grimm J.B., et al. (2021) A general method to improve fluorophores using deuterated auxochromes. JACS Au. 1, 690–6.

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