Determination of ROS Generated by Arabidopsis Xanthine Dehydrogenase1 (AtXDH1) Using Nitroblue Tetrazolium (NBT) and 3,3′-Diaminobenzidine (DAP)

Aigerim Soltabayeva, Moshe Sagi

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Plants generate reactive oxygen species (ROS) during different metabolic processes, which play an essential role in coordinating growth and response. ROS levels are sensitive to environmental stresses and are often used as a marker for stress in plants. While various methods can detect ROS changes, histochemical staining with nitroblue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB) is a popular method, though it has faced criticism. This staining method is advantageous as it enables both the quantification and localization of ROS and the identification of the enzymatic origin of ROS in plants, cellular compartments, or gels. In this protocol, we describe the use of NBT and DAP staining to detect ROS generation under different stresses such as nitrogen starvation, wounding, or UV-C. Additionally, we describe the use of NBT staining for detecting enzymatic generation of ROS in native and native SDS PAGE gels. Our protocol also outlines the separation and comparison of the origin of ROS generated by xanthine dehydrogenase1 (XDH1) using different substrates.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages65-77
Number of pages13
DOIs
StatePublished - 1 Jan 2024

Publication series

NameMethods in Molecular Biology
Volume2798
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Arabidopsis
  • NBT and DAP staining
  • Native and native-SDS PAGE in gel assay
  • Nitrogen starvation
  • ROS
  • Wounding
  • XDH1
  • or UV-C

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

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