Nitric Oxide pp 205-221 | Cite as

Detection of S-Nitrosated Nuclear Proteins in Pathogen-Treated Arabidopsis Cell Cultures Using Biotin Switch Technique

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)

Abstract

Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193–197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively.

Key words

Nitric oxide S-Nitrosation Biotin switch assay Suspension cell culture Nuclear proteins Pseudomonas syringae Arabidopsis thaliana 

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Iranian Research Institute of Plant ProtectionAgricultural Research, Education and Extension Organization (AREEO)TehranIran
  2. 2.Helmholtz Zentrum München–German Research GmbH, Center for Environment HealthInstitute of Biochemical Plant Pathology, Ingolstädter Landstraße 1Munich-NeuherbergGermany

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