CY5-N3 (Synonyms: Sulfo-Cyanine5-azide)

Live Cell-Based Fluorescent Labeling and Imaging of Target Proteins with CY5-N3^[1]

This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.

1. Probe Incubation: Prepare a stock solution of the designed and synthesized small molecule photoaffinity probe by dissolving it in DMSO. For the cell imaging experiment, incubate HeLa cells with the probe (typically at a working concentration of 2μM) in complete DMEM medium at 37°C with 5% CO₂ for 2 hours. Ensure the final concentration of DMSO in the medium does not exceed 1% during incubation.

2. Photo-crosslinking and Cell Fixation: After incubation, aspirate and discard the medium. Wash the cells twice with DMEM and twice with PBS to remove excess unbound probe. Place the cells on ice and irradiate with UV light at a wavelength of 365nm for 15-20 minutes. This activates the photoreactive group (e.g., an aliphatic diazirine) on the probe, enabling covalent crosslinking with target proteins. Subsequently, fix the cells with DMEM containing 3.7% paraformaldehyde at room temperature for 30 minutes.

3. Cell Permeabilization and Blocking: Discard the fixative and wash the cells twice with PBS. Treat the cells with PBS containing 0.1% Triton X-100 at room temperature for 10 minutes to increase cell membrane permeability. After permeabilization, wash the cells twice with PBS, then block with PBS containing 2% BSA and 0.05% Tween-20 at room temperature for 30 minutes to reduce non-specific binding.

4. Click Chemistry Labeling: Discard the blocking solution and wash the cells twice with PBS containing 0.05% Tween-20. Add freshly prepared click chemistry reaction solution to the cells. The final composition of the reaction solution is: CY5-N3 (5μM), TBTA (20μM), TCEP (200μM), and CuSO₄ (200μM) dissolved in PBS, with a total volume of 100μL. Allow the reaction to proceed at room temperature, protected from light, for 2 hours. This copper-catalyzed azide-alkyne cycloaddition (CuAAC) covalently links the CY5 fluorescent dye to the alkyne group on the probe, which is already crosslinked to the target protein.

5. Washing and Nuclear Staining: After the reaction, sequentially wash the cells 3 times (10 minutes each) with PBS containing 0.05% Tween-20 and 0.5 mM EDTA, followed by 2 washes with PBS (5 minutes each). To stain the nuclei, incubate with PBS containing 0.25mg/mL Hoechst 33258 at room temperature, protected from light, for 15 minutes. Finally, wash once with PBS and once with deionized water. The cells are then ready for mounting and imaging.

6. Image Acquisition: Use a confocal microscope (e.g., Zeiss LSM 510 Meta or Leica TCS SP5X system) to acquire fluorescence images in the CY5 channel (ex/em=646/662nm) and the Hoechst channel (for nuclei) at their respective excitation/emission wavelengths.

Precautions:

(1) The CY5-N3 reagent is light-sensitive. All operations should be performed protected from light.

(2) For your safety and health, please wear a lab coat and disposable gloves during operation.

References:
[1] Su Y, Pan S, Li Z, et al. Multiplex imaging and cellular target identification of kinase inhibitors via an affinity-based proteome profiling approach. Sci Rep. 2015 Jan 12;5:7724.