Identification of Brassinosteroid Signaling Complexes by Coimmunoprecipitation and Mass Spectrometry
Walter van Dongen1, Luc van Heerde1, Sjef Boeren1
1Laboratory of Biochemistry, Wageningen UR, 6708, WE, Wageningen, The Netherlands.
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.
More Related Videos
09:02Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment
Published on: April 20, 2018
14:58Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
Published on: November 12, 2012
Related Concept Videos
Assembly of Signaling Complexes
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...
Cell Signaling in Plants
Peptide Identification Using Tandem Mass Spectrometry
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
Protein Networks
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
