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.
