Cy3 methyltetrazine (TZ-Cy3) is aclick chemistryreagent withmethyltetrazinebuilding blocks that is highly reactive towardscyclooctene.Cy3 methyltetrazine is also atetrazine-modified fluorescent probethat can be used to analyze protein phosphorylation in solution and living cells^[1].

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) for1 h.
3. Add Cy3 methyltetrazine to label the phosphorylated protein with fluorescence.
4. Incubate with fresh medium for1 h.
5. Wash the cells twice with PBS to release the free Cy3 methyltetrazine probe.
6.Incubate the cells with 4% paraformaldehyde for15 minand with0.1%Triton X-100 solution at room temperature for10 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).

727.64

C₄₀H₄₆BF₄N₇O

2183473-57-4

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.