SNAP-TMR

Guidelines (The following is a recommended experimental protocol for reference only, and adjustments should be made according to specific requirements)
1. Stock Solution Preparation
1.1 Solvent: DMSO; alternatively, DMEM can be used.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution at 1-10 mM.
2. Working Solution Preparation
2.1 Diluent: Cell lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween20 (HY-141415), 1× protease inhibitor cocktail); alternatively, DMEM can be used.
2.2 Working Concentration: 1.5 μM; alternatively, 3 μM can be used.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use the solution immediately.
3. Staining Procedures
3.1 Applicable to adherent and suspension cells expressing SNAP fusion proteins^[1]:
3.1.1 Sample Preparation: Collect adherent cells after trypsinization; collect suspension cells by centrifugation at 1000 g for 5 min at room temperature.
3.1.2 Endogenous SNAP-tagged cell lines (e.g., HAP1 SNAP-CLCaEN) are also applicable.
3.1.3 Washing Before Labeling: Wash the collected cells twice with PBS before lysis for labeling.
3.1.4 Incubation Conditions: Incubate the cell lysis buffer with 1.5 μM SNAP-TMR working solution at room temperature for 30 min.
3.2 Applicable to adherent mammalian cells (COS7 cells)^[2]:
3.2.1 Incubation Conditions: Incubate the cells with 3 μM SNAP-TMR working solution at 37°C under 5% (vol/vol) CO₂ for 30 min.
3.2.2 Cell Collection: Collect cells by trypsinization after incubation.
3.2.3 Washing Steps: Wash the cells three times with DMEM, followed by incubation in DMEM for 30 min to remove unbound ligands.
4. Detection and Analysis
4.1 Instrument Type: Gel imaging system; alternatively, total internal reflection fluorescence (TIRF) microscope can be used.
4.2 Excitation/Emission Wavelength: Consistent with Cy3/TMR spectra; alternatively, use 561 nm excitation and TMR emission.
4.3 Result Analysis:
4.3.1 Fluorescence Properties: SNAP-TMR produces red/purple or red fluorescence, and the signal intensity is sufficient to support single-molecule detection.
4.3.2 Localization: Fluorescence signals localize to the subcellular compartments of SNAP fusion proteins (e.g., clathrin-coated vesicles corresponding to SNAP-CLCaEN, kinesin-1 motors distributed along microtubule tracks).
4.3.3 Quantitative Analysis: Quantify the fluorescence intensity of SNAP-TMR-labeled SNAP fusion proteins with reference to a total protein loading control (e.g., TCE staining).
4.3.4 Motility Analysis (Applicable to samples labeled with motor proteins): Generate kymographs to measure the run length, dwell time and velocity of labeled motors; analyze the cumulative distribution function to determine the mean values.

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