Identification of brassinosteroid signaling complexes by coimmunoprecipitation and mass spectrometry

Walter van Dongen*, Luc van Heerde, Sjef Boeren, Sacco C. de Vries

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Citations (Scopus)

Abstract

A combination of coimmunoprecipitation (coIP) of tagged proteins followed by protein identification and quantitation using Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) has proven to be a reliable method to qualitatively characterize membrane-bound receptor complexes from plants. Success depends on a range of parameters, such as abundance and stability of the complex and functionality of the tagged receptors, efficiency of the protein complex isolation procedure, MS equipment, and analysis software in use. In this Chapter, we focus on the use of one of the green fluorescent protein-tagged receptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, of which SERK3, also known as BRASSINOSTEROID INSENSITIVE1 (BRI1) ASSOCIATED KINASE1 (BAK1), is a coreceptor of BRI1. Like BRI1 itself, SERK3 is a leucine-rich repeat receptor kinase (LRR RK) with a single-pass transmembrane domain. The latest updated laboratory protocol is presented as well as examples of data analysis and typical results obtained. Potential drawbacks of the procedure employed for plant membrane proteins will be pointed out.

Original languageEnglish
Title of host publicationBrassino-steroids
EditorsEugenia Russinova, Ana I. Caño-Delgado
PublisherHumana Press Inc.
Pages145-154
Number of pages10
ISBN (Electronic)9781493968138
ISBN (Print)9781493968114
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1564
ISSN (Print)1064-3745

Keywords

  • Arabidopsis
  • GFP
  • Immunoprecipitation
  • Protein complex identification
  • Protein–protein interaction
  • Tandem mass spectrometry

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    van Dongen, W., van Heerde, L., Boeren, S., & de Vries, S. C. (2017). Identification of brassinosteroid signaling complexes by coimmunoprecipitation and mass spectrometry. In E. Russinova, & A. I. Caño-Delgado (Eds.), Brassino-steroids (pp. 145-154). (Methods in Molecular Biology; Vol. 1564). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6813-8_12