Cy3 methyltetrazine  (Synonyms: TZ-Cy3)

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].

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).

727.64

C₄₀H₄₆BF₄N₇O

2183473-57-4

Solid

Brown to reddish brown

570

550

O=C(CCCCC[N+]1=C(/C=C/C=C2N(C)C3=C(C\2(C)C)C=CC=C3)C(C)(C)C4=C1C=CC=C4)NCC5=CC=C(C6=NN=C(C)N=N6)C=C5.[F-][B+3]([F-])([F-])[F-]

Room temperature in continental US; may vary elsewhere.

-20°C, sealed storage, away from moisture and light

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Li Y, et al. Analysis of protein phosphorylation in solution and in cells by using an ATP analogue in combination with fluorescence techniques. Analyst. 2021 Jul 21;146(14):4506-4514.  [Content Brief]