Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 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.

PubMed Disclaimer

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

Similar articles

See all similar articles

References

    1. Speers AE, Adam GC, Cravatt BF. Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition. J Am Chem Soc. 2003;125:4686–4687. - PubMed
    1. Peng T, Thinon E, Hang HC. Proteomic analysis of fatty-acylated proteins. Curr Opin Chem Biol. 2016;30:77–86. - PMC - PubMed
    1. Tuladhar R, Lum L. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making. Biochem Soc Trans. 2015;43:235–239. - PMC - PubMed
    1. Dodge ME, Moon J, Tuladhar R, Lu J, Jacob LS, Zhang LS, Shi H, Wang X, Moro E, Mongera A, Argenton F, Karner CM, Carroll TJ, Chen C, Amatruda JF, Lum L. Diverse chemical scaffolds support direct inhibition of the membrane-bound O-acyltransferase porcupine. J Biol Chem. 2012;287:23246–23254. - PMC - PubMed
    1. Herr P, Basler K. Porcupine-mediated lipidation is required for Wnt recognition by Wls. Dev Biol. 2012;361:392–402. - PubMed

Publication types

LinkOut - more resources