Halo tag TMR (solution)

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
Staining Procedure^[1] :
1. Transfect the cells with the plasmid encoding the HaloTag fusion protein, and culture them under appropriate conditions until reaching the desired confluence (recommend 70-80%, to avoid excessive fusion which may affect the staining efficiency).
2. Prepare 5× HaloTag TMR working solution (final concentration 25 μM, diluted from the stock solution using serum-free medium or PBS) in advance; Remove 1/5 of the old medium, add an equal volume of 5× working solution, so that the final dye concentration in the system is 5 μM. Gently shake the culture vessel to mix evenly (or directly replace with pre-heated serum-free medium containing 5 μM TMR to avoid excessive local concentration).
3. Place the cells in a 37°C, 5% CO? incubator under light conditions for 15 minutes to allow the ligand to undergo covalent binding with the HaloTag protein.
4. For the membrane-permeable ligands: Remove the medium containing the ligands, replace it with an equal volume of pre-warmed (to 37°C) serum-free medium or PBS. Repeat this washing step twice, and perform a total of three washes. Be gentle during the process to avoid cell detachment. For cell surface markers (non-membrane-permeable ligands): Wash twice with pre-warmed serum-free medium or PBS to remove excess ligands from the extracellular environment.
5. The cells were further incubated in a 37°C complete medium in the dark for 30 minutes to ensure that the unbound ligands in the cells were actively excreted/metabolized, thereby significantly reducing the background fluorescence.
6. Replace the culture medium with a phenol red-free one (if available) to reduce autofluorescence, and avoid direct exposure to strong light throughout the process.
7. Transfer the cells to a container suitable for imaging. Observe them using a fluorescence microscope (TMR imaging recommends an excitation wavelength of 555 nm and an emission wavelength of 580 nm. Redox Red/ Texas Red filters can be used).

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