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Methods Mol Biol ; 1564: 145-154, 2017.
Article in English | MEDLINE | ID: mdl-28124252

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.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Gene Expression Regulation, Plant , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Seedlings/genetics , Signal Transduction , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Brassinosteroids/metabolism , Chromatography, Liquid , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunoprecipitation/methods , Molecular Sequence Annotation , Plants, Genetically Modified , Protein Binding , Protein Interaction Mapping , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Seedlings/metabolism , Tandem Mass Spectrometry/methods
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