Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction
We describe here a technique for visualizing the lipidation status of Wnt proteins using azide-alkyne cycloaddition chemistry (click chemistry) and SDS-PAGE. This protocol incorporates in vivo labeling of a Wnt-IgG Fc fusion protein using an alkynylated palmitate probe but departs from a traditional approach by incorporating a secondary cycloaddition reaction performed on single-step purified Wnt protein immobilized on protein A resin. This approach mitigates experimental noise by decreasing the contribution of labeling from other palmitoylated proteins and by providing a robust method for normalizing labeling efficiency based on protein abundance.
Key wordsWnt acylation Click chemistry Biotinylation Palmitoleate IgG fusion protein
This work was supported by the NIH (R01 CA168761, R01CA196851), CPRIT (RP130212), and the Welch Foundation (I-1665).
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