Halo tag TMR

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
Staining steps^[1]:
1) Transfect cells with a plasmid encoding the target HaloTag fusion protein and culture to the desired confluence under appropriate conditions.
2) Replace 1/5 of the serum-free culture medium or PBS with 5× HaloTag TMR working solution to a final concentration of 5 μM. Mix by gently shaking the culture container.
3) Incubate the cells in a 37°C, CO₂ incubator for 15 minutes to allow the ligand to covalently bind to the HaloTag protein.
4) For permeable ligands: Remove the ligand-containing medium and replace with an equal volume of prewarmed serum-free culture medium or PBS. Repeat this wash step two more times for a total of three washes. For cell surface markers (non-permeable ligands): Wash twice with serum-free culture medium or PBS to remove excess ligand from the extracellular environment.
5) Incubate the cells in complete medium at 37°C for an additional 30 minutes to ensure complete removal of unbound ligand.
6) Replace the medium with phenol red-free medium (if available) to reduce autofluorescence.
7) Transfer the cells to a container suitable for imaging and observe using a fluorescence microscope.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.