Palmitic Acid Alkyne

Click palmitoylation assay

1.Seed HEK293T cells, and transfect cells with plasmids (YKT6-WT BioID, YKT6-3E BioID and Wnt3A) in DMEM supplemented with 10% FBS.

2.Dissolve ω-Alkynyl palmitic acid (Palmitic Acid Alkyne) in ethanol to a final concentration of 50mM and stored at −80°C.

3.Dilute Palmitic acid alkyne to a final concentration of 100μM in DMEM supplemented with 5% FBS (fatty acid-free) sonicated for 15min at room temperature in a water bath and then allowed to precomplex for another 15min.

4.Add Palmitic acid alkyne-containing medium was added and replace partially after 24h.

5.At 72h post-transfection, lysis cells (PBS with 1% Triton x-100, 0.1% SDS, PIC), then centrifuge at 16,000g for 5min at 4°C.

6.Precipitate lysates with Wessel-Flugge Protein precipitation.

7.Dissolve the click labelling reaction [0.1mM biotin-azide, 1mM Tris(2-carboxyethyl)phosphine hydrochloride] in water

8.Incubate with 0.1mM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine dissolved in DMSO and 1mM CuSO₄ in water by shaking for 2h at 37°C under dark conditions.

9.After the click reaction, precipitate the samples were with 10× methanol overnight at −80°C, then centrifuge and wash again with ice-cold methanol

10.Resuspend the dried pellet in 4% SDS.

11.Precipitate click-biotinylated proteins with High Capacity Neutravidin Agarose Resin .

12.Wash samples with 1% SDS and eluted, then analysis further by immunoblotting.

*This protocol only provides a guideline, and should be modified according to your specific needs

References:
1.Linnemannstöns, Karen et al. “Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium.” Development (Cambridge, England) vol. 147,15 dev185421. 14 Aug. 2020, doi:10.1242/dev.185421