Th-TMR-S-Halo

Operating Instructions
Operating Instructions (The following is a recommended experimental protocol for guidance only, and adjustments are required based on specific needs).

1. Stock Solution Preparation
1.1 Solvent: For most dyes, organic solvents are commonly used for dissolution, such as anhydrous DMSO.
1.2 Concentration Recommendation: It is recommended to prepare a high-concentration stock solution at 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: 20 mM HEPES buffer, 150 mM NaCl, pH 7.4.
2.2 Working Concentration: 500 nM, 10 nM.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedures
3.1 Sample Types: Adherent cells (MCF7, HEK293T, U2OS, COS7)
3.1.1 Adherent cells: Trypsin digestion is not required for live cell imaging.
3.2 Incubation Conditions:
3.2.1 Live cell nucleus labeling: 500 nM.
3.2.2 Live cell endoplasmic reticulum (ER) labeling: Incubate with 500 nM working solution for 60 min.
3.2.3 Fixed cell tubulin labeling: Incubate with 10 nM working solution.
3.3 Washing Procedures:
3.3.1 ER labeling: Wash with HBSS buffer after incubation.
3.3.2 Wash-free imaging: No washing step is required.

4. Control Setup
4.1 Non-transfected cells serve as negative controls to verify target-specific labeling.
4.2 DMSO-treated cells serve as controls for endoplasmic reticulum stress experiments.

5. Detection and Analysis
5.1 Instruments: Confocal fluorescence microscope, super-resolution microscope (NSPARC, SIM).
5.1.1 Excitation wavelength (Ex): 561 nm; Emission wavelength (Em): 570−616 nm for live cell super-resolution imaging, 580−610 nm for confocal imaging, and 620 nm for fixed cell super-resolution imaging.
5.2 Result Analysis:
5.2.1 Fluorescence intensity change: The red fluorescence is significantly enhanced after Th-TMR-S-Halo binds to HaloTag. The fluorescence quantum yields (Φ) of Th-TMR-S-Halo in the free state and HaloTag-bound state are 0.01 and 0.44, respectively.
5.2.2 Fluorescence localization: Cells expressing Halo-NLS show nuclear localization; cells expressing Halo-Sec61β show ER localization; cells expressing tubulin-Halo show cytoskeleton (tubulin) localization.
5.2.3 Color change: Red fluorescence.

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