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.
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Acknowledgement
This work was supported by the NIH (R01 CA168761, R01CA196851), CPRIT (RP130212), and the Welch Foundation (I-1665).
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Tuladhar, R., Yarravarapu, N., Lum, L. (2016). Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction. In: Barrett, Q., Lum, L. (eds) Wnt Signaling. Methods in Molecular Biology, vol 1481. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6393-5_2
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DOI: https://doi.org/10.1007/978-1-4939-6393-5_2
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6391-1
Online ISBN: 978-1-4939-6393-5
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