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 - Jan 1 2016

Publication series

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

Fingerprint

Wnt Proteins
Acylation
Cycloaddition Reaction
Click Chemistry
Immobilized Proteins
Proteins
Alkynes
Azides
Palmitates
Staphylococcal Protein A
Noise
Polyacrylamide Gel Electrophoresis
Immunoglobulin G
In Vitro Techniques

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Tuladhar, R., Yarravarapu, N., & Lum, L. (2016). Monitoring wnt protein acylation using an in vitro cyclo- addition reaction. In Methods in Molecular Biology (pp. 11-16). (Methods in Molecular Biology; Vol. 1481). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6393-5_2

Monitoring wnt protein acylation using an in vitro cyclo- addition reaction. / Tuladhar, Rubina; Yarravarapu, Nageswari; Lum, Lawrence.

Methods in Molecular Biology. Humana Press Inc., 2016. p. 11-16 (Methods in Molecular Biology; Vol. 1481).

Research output: Chapter in Book/Report/Conference proceedingChapter

Tuladhar, R, Yarravarapu, N & Lum, L 2016, Monitoring wnt protein acylation using an in vitro cyclo- addition reaction. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1481, Humana Press Inc., pp. 11-16. https://doi.org/10.1007/978-1-4939-6393-5_2
Tuladhar R, Yarravarapu N, Lum L. Monitoring wnt protein acylation using an in vitro cyclo- addition reaction. In Methods in Molecular Biology. Humana Press Inc. 2016. p. 11-16. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-6393-5_2
Tuladhar, Rubina ; Yarravarapu, Nageswari ; Lum, Lawrence. / Monitoring wnt protein acylation using an in vitro cyclo- addition reaction. Methods in Molecular Biology. Humana Press Inc., 2016. pp. 11-16 (Methods in Molecular Biology).
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