Promega HT1010 JFX HaloTag Ligands Instructions

Promega HT1010 JFX HaloTag Ligands

Product Information

• Specifications:
  • Models: HT1010, HT1020, HT1030, HT1040, HT1050, HT1060, HT1070, HT1100, HT1110
  • Ligand Excitation Maximum: 503nm, 549nm, 554nm, 585nm, 635nm, 646nm, 650nm
  • Ligand Emission Maximum: 529nm, 571nm, 576nm, 609nm, 652nm, 664nm, 667nm

Product Usage Instructions

• Materials Required:
  • Cells
  • Cell medium or aqueous buffer
  • 5X working stock solution
  • Phenol red-free medium (optional)
  • Microscope
• Protocol:
  • Prepare the cells as per standard laboratory protocols.
  • Add 1 ml of cell medium or the chosen aqueous buffer to the cells.
  • Incubate the cells under appropriate conditions.
  • Add the 5X working stock solution to cells at a 1X final concentration of 200nM.
  • Optimize ligand concentration if necessary.
  • Aspirate medium and replace with fresh cell medium or phenol red-free medium.
  • Transfer the cells to a microscope for imaging.
• Ligand Information:
  • For optimal results, refer to the ligand excitation and emission maximum values provided.
  • Adjust ligand concentrations based on cell expression levels for a better signal-to-background ratio.
• Additional Resources:
  • For more detailed protocol information, refer to Technical Manual #TM260 available online at: www.promega.com

FAQs

• Q: What should I do if I observe low signal intensity?
  • A: Try increasing the ligand concentration or extending the incubation time to enhance signal intensity.
• Q: Can I use a different type of buffer instead of the recommended cell medium?
  • A: It is recommended to use the specified cell medium or an aqueous buffer for optimal results.

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

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

Protocol

1. 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 or vortex 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.

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.

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.

CONTACT

• PROMEGA CORPORATION
• 2800 WOODS HOLLOW ROAD
• MADISON, WI 53711-5399 USA
• TELEPHONE 608-274-4330
• WWW.PROMEGA.COM.
• ©2023 PROMEGA CORPORATION
• ALL RIGHTS RESERVED
• PRICES AND SPECIFICATIONS SUBJECT TO CHANGE WITHOUT PRIOR NOTICE
• 11/23
• PART# FB248
• Instructions for Use of Products
  • HT1010,
  • HT1020,
  • HT1030,
  • HT1040,
  • HT1050,
  • HT1060,
  • HT1070,
  • HT1100 and HT1110.

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