Cy3 methyltetrazine

Cy3 methyltetrazine (TZ-Cy3) is a click chemistry reagent with methyltetrazine building blocks that is highly reactive towards cyclooctene. Cy3 methyltetrazine is also a tetrazine-modified fluorescent probe that can be used to analyze protein phosphorylation in solution and living cells[1].

Research Areas >> Others

Signaling Pathways >> Others >> Others

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). Confocal imaging of phosphorylation in cells: 1. Culture H1299 cells in a coverglass-bottom imaging dish for 24 h (take H1299 cells as an example). 2. Incubate H1299 cells with different concentrations of ATP-NB (a ATP analogue functionalized by norbornene, which can penetrate cells and efficiently phosphorylate proteins in living cells) for 1 h. 3. Add Cy3 methyltetrazine to label the phosphorylated protein with fluorescence. 4. Incubate with fresh medium for 1 h. 5. Wash the cells twice with PBS to release the free Cy3 methyltetrazine probe. 6.Incubate the cells with 4% paraformaldehyde for 15 min and with 0.1% Triton X-100 solution at room temperature for 10 min. 7. Wash cells 4 times with PBS. 8. Use a confocal microscopy imaging system to obtain the high-resolution images of cells (Ex=488 nm; Em=561 nm).