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Curr Protoc Chem Biol. 2017 Jun 19;9(2):117-127. doi: 10.1002/cpch.20.

Local Generation and Imaging of Hydrogen Peroxide in Living Cells.

Author information

1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia.
2
EMBL Heidelberg, Heidelberg, Germany.
3
Oregon Health & Science University, Portland, Oregon.
4
Institute for Cardiovascular Physiology, Georg August University Göttingen, Göttingen, Germany.

Abstract

Described here is a localized H2 O2 generation-detection system consisting of a yeast D-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H2 O2 dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H2 O2 generated by the activated DAAO. The system can be easily optimized for various combinations of cell types, conditions and DAAO/sensor subcellular localizations.

KEYWORDS:

D-amino acid oxidase; HyPer; HyPer2; HyPerRed; biosensors; hydrogen peroxide; metabolic engineering; reactive oxygen species

PMID:
28628200
DOI:
10.1002/cpch.20
[Indexed for MEDLINE]

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