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. 2016:1481:11-6.
doi: 10.1007/978-1-4939-6393-5_2.

Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction

Rubina Tuladhar  1 , Nageswari Yarravarapu  1 , Lawrence Lum  2
Affiliations

Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction

Rubina Tuladhar et al. Methods Mol Biol. 2016.

Abstract

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.

Keywords: Biotinylation; Click chemistry; IgG fusion protein; Palmitoleate; Wnt acylation.

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Figures

Fig. 1
Fig. 1
Overview of modified Wnt labeling protocol using an alkynylated fatty acyl probe and copper-catalyzed alkyne-azide cycloaddition
See this image and copyright information in PMC

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