Monitoring wnt protein acylation using an in vitro cyclo- addition reaction

Rubina Tuladhar, Nageswari Yarravarapu, Lawrence Lum

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages11-16
Number of pages6
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1481
ISSN (Print)1064-3745

Keywords

  • Biotinylation
  • Click chemistry
  • IgG fusion protein
  • Palmitoleate
  • Wnt acylation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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