4-Azido-L-phenylalanine (p-Azidophenylalanine)

This protocol only provides a guideline, and should be modified according to your needs.
Unnatural amino acid labeling in living cells via azide-alkyne click chemistry^[1].
1. Cell culture:
At 24h before transfection, HEK293T cells were cultured on 18mm poly-lysine-coated glass coverslips, the cell culture medium was changed to supplemented with 0.65mM 4-Azido-L-phenylalanine (p-Azidophenylalanine). growth medium.
2. Transfection experiments
pIRE4-Azi plasmid (1:1 w/w) was transfected into cells using Lipofectamine 3000 reagent. After 24h, the growth medium was refreshed with 0.65mM 4-Azido-L-phenylalanine (p-Azidophenylalanine) and the cells were cultured for an additional 24h (48h total expression). 30min before labeling, 4-azido-L-phenylalanine supplemented growth medium was removed, and cells were washed twice by extracellular buffer solution containing 128mM NaCl, 2mM KCl, 2.5mM CaCl₂, 1.2mM MgCl₂, 10mM sucrose, 10mM HEPES, and pH7.4 and were kept in growth medium without 4-azido-L-phenylalanine.
3. Unnatural amino acid labeling in living cells via azide-alkyne click chemistry
3.1 Preparation of stock solutions
Sulfo-Cyanine3.5 alkyne (catalog number: GC59209) alkyne dyes 10mM in DMSO, BTTES (Click Chemistry Tools) 50mM, copper sulfate (catalog number: GB57835) 20mM, Aminoguanidine (catalog number: GC15727) 100mM, and (+)-sodium L-ascorbate (catalog number: GC13616) 100mM in ultrapure distilled water.
3.2 Click on Chemical Reaction
A: In 656µl of extracellular buffer solution, Cy3.5 were mixed to a final concentration of 20µM. Fresh copper sulfate solution and BTTES (1:5 molar ratio) solution were premixed and then added to a final concentration of 150µM. Next, add Aminoguanidine to a final concentrate. The total labeling volume is 0.7mL;
B: Incubate the completed labeling mixture at 4°C for 8 minutes, then at room temperature for 2 minutes and in the dark before adding to the cells. Wash the cells before adding the labeling mixture. During labeling, keep the cells in the dark at 37°C and 5% CO₂ for 10-15 minutes. After labeling, wash the cells twice with extracellular buffer. Cy3.5-labeled products can be observed at 591/604nm using confocal microscopy.
References:
[1].Schamber M R, Vafabakhsh R. Mechanism of sensitivity modulation in the calcium-sensing receptor via electrostatic tuning[J]. Nature communications, 2022, 13(1): 2194.