Determination of Protein S-Acylation State by Enhanced Acyl-Switch Methods

Charlotte H. Hurst, Dionne Turnbull, Piers A. Hemsley (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

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Abstract

S-Acylation is increasingly being recognized as an important dynamic posttranslational modification of cysteine residues in proteins. Various approaches have been described for assaying protein S-acylation with acyl-switch approaches being the most common and accessible. However, these approaches can be time-consuming with low reproducibility as a result of multiple protein precipitation/resuspension cleanup steps. Here we present a faster, cleaner, and more sensitive acyl-switch approach for detecting the S-acylation state of any protein, from any cell or tissue type, that can be detected by western blotting. In the case of acyl-RAC, the procedure is now performed without protein precipitation, greatly increasing speed and improving sample handling in the assay. This also allows for more samples to be processed simultaneously and opens the way for medium-throughput assays. Overall, maleimide scavenging improves the reliability of determination and quantification of protein S-acylation state by acyl-switch methods.

Original languageEnglish
Title of host publicationProtein Lipidation
Subtitle of host publicationMethods and Protocols
EditorsMaurine E. Linder
Place of PublicationNew York
PublisherHumana Press
Chapter1
Pages3-11
Number of pages9
Volume2009
ISBN (Electronic)9781493995325
ISBN (Print)9781493995318
DOIs
Publication statusPublished - 2019

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • 2,3-Dimethyl 1,3-butadiene
  • Acyl-switch
  • Biotin
  • Diels–Alder
  • Maleimide
  • N-Ethylmaleimide
  • Palmitoylated
  • S-Acylated
  • S-Acylation
  • S-Palmitoylation
  • Diels-Alder
  • 2,3-Dimethyl 1,3-butadiene,Biotin

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  • Cite this

    Hurst, C. H., Turnbull, D., & Hemsley, P. A. (2019). Determination of Protein S-Acylation State by Enhanced Acyl-Switch Methods. In M. E. Linder (Ed.), Protein Lipidation: Methods and Protocols (Vol. 2009, pp. 3-11). (Methods in Molecular Biology). Humana Press. https://doi.org/10.1007/978-1-4939-9532-5_1