Alkyne tyramide (Synonyms: Alk-Ph)

This experiment comes from the literature, please modify it according to your specific needs.

1. The yeast strain transformation was grown in SD cell culture to the logarithmic metaphase and then inoculated into SG cell medium or YPG medium. The cells were collected and washed twice with PBS buffer.

2. Cell precipitates were re-suspended in PBS buffer containing 2.5mM Alkyne tyramide (about 500μL Alkyne tyramide solution for 5 OD₆₀₀ yeast cells) and incubated at room temperature for 30min. Then H₂O₂ was added for 1min to trigger the production of phenol free radicals and APEX2 labeling.

3. Add an equal volume solution of quench agent (10mM sodium azide, 10mM sodium ascorbate and 5mM trolox) to stop Alkyne tyramide labeling.

4. After APEX2 labeling, cells were washed twice with PBS buffer and then re-suspended in PBS buffer with protease inhibitor cocktail.

5. The cells were mixed with equal-volume glass beads and vibrated at 2000rpm at 4°C for 1 minute. After that, the cells were placed on ice to cool for 1 minute. Repeat the shake-cold cycle 10 times to lyse the yeast cells. Excess small molecules are removed by precipitating the protein in cold methanol at -80°C for 3 hours.

6. The protein was precipitated and suspended in 0.5% SDS water, then in the presence of CuSO₄ (333μM), BTTAA (666μM) and sodium ascorbate (2.5mM), Reaction with azide Cy5 (100μM) at room temperature for 1h. Labeled protein samples were separated on 12% SDS-PAGE gel.

7. For fluorescence analysis in the gel, the gel was rinsed withdestaining solution (60% v/v water, 30% v/v methanol, 10% v/v acetic acid), and then the imaging was carried out on the Typhoon FLA 9500 imager.

References:

[1] Li Y, Tian C, Liu K, et al. A clickable APEX probe for proximity-dependent proteomic profiling in yeast[J]. Cell Chemical Biology, 2020, 27(7): 858-865. e8.