Th-TMR-S-SNAP

Operating Instructions (The following is a recommended experimental protocol for guidance only, and adjustments should be made according to your specific requirements).
1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration Recommendation: Prepare an appropriate concentration of stock solution of Th-TMR-S-SNAP with DMSO, and store it at -20°C in the dark.
2. Working Solution Preparation
2.1 Diluent: 20 mM HEPES buffer, 150 mM NaCl, pH 7.4.
2.2 Working Concentrations: 500 nM; 10 nM.
2.3 Precautions: Adjust the concentration of the working solution as needed; prepare and use it immediately.
3. Staining Procedure
3.1 Sample Type: Adherent cells^[1]
3.1.1 Adherent cells: Trypsin digestion is not required for live cell imaging.
3.2 Incubation Conditions: No-wash condition is adopted for live cell imaging, with incubation in 500 nM working solution for 60 min; fixed cell imaging is performed with incubation in 10 nM working solution.
3.3 Washing Step: Wash with HBSS buffer after incubation for fixed cell imaging.
4. Control Setup
4.1 Use untransfected cells as the negative control.
5. Detection and Analysis
5.1 Instruments: Confocal fluorescence microscope; Super-resolution structured illumination microscope (SIM)
5.1.1 Excitation/emission wavelengths: For live cell super-resolution imaging, λex = 561 nm; λem = 570−616 nm; for fixed cell SIM imaging, λex = 561 nm; λem = 620 nm.
5.2 Result Analysis:
5.2.1 Fluorescence intensity change: The nuclear fluorescence intensity of cells expressing SNAP-tag is 16.2 times higher than that of the cytoplasm in untransfected cells.
5.2.2 Fluorescence localization: SNAP-tag labels inside cells are localized in the nucleus; SNAP-tag labels on the cell surface are localized in the cell membrane.
5.2.3 Color change: Red fluorescence is emitted.