Related Experiment Video
Updated: Mar 17, 2026
09:47
Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis
Published on: June 2, 2023
3.5K
Quantification of hydrogen peroxide in plant tissues using Amplex Red
Sourav Chakraborty1, Amy L Hill1, Gautam Shirsekar1
1Department of Plant Pathology, The Ohio State University, 2021 Coffey Road, 201 Kottman Hall, Columbus, OH 43210, USA.
Methods (San Diego, Calif.)
|August 1, 2016
Summary
This study optimized an Amplex Red assay for accurately measuring hydrogen peroxide (H2O2) in plant tissues. The improved method overcomes interference from common extraction additives, ensuring reliable H2O2 quantification for plant research.
Area of Science:
- Plant Physiology
- Biochemistry
- Molecular Biology
Background:
- Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are crucial signaling molecules in plant stress responses and development.
- Accurate quantification of H2O2 in plant tissues is challenging due to its instability and interference from extraction procedures.
- H2O2 plays a vital role in plant defense signaling and programmed cell death.
Purpose of the Study:
- To optimize and validate an Amplex Red-based assay for reliable H2O2 quantification in plant tissue lysates.
- To assess the impact of common extraction additives on H2O2 stability and assay accuracy.
- To determine the stability of the optimized Amplex Red working solution for long-term storage.
Main Methods:
- Optimization of an Amplex Red assay for H2O2 estimation in plant tissue extracts.
- Evaluation of various extraction additives (e.g., activated charcoal, PVPP, TCA) for interference and degradation effects.
- Quantification of constitutive and induced H2O2 levels in *Pinus nigra*, *Oryza sativa*, and *Arabidopsis thaliana*.
- Assessment of Amplex Red working solution stability over one month at -80°C.
Main Results:
- The optimized Amplex Red assay demonstrated limits of detection and quantitation at 6 and 18 picomoles, respectively.
- Several commonly used extraction additives were found to degrade H2O2 or interfere with the Amplex Red assay.
- The assay achieved sensitivity in the nmol g⁻¹ FW range for H2O2 quantification in model plants.
- The Amplex Red working solution remained significantly stable when stored at -80°C for one month.
Conclusions:
- The optimized Amplex Red assay provides a reliable and sensitive method for quantifying H2O2 in diverse plant tissues.
- Careful selection of extraction methods is critical to avoid H2O2 degradation and assay interference.
- This validated method facilitates further research into the role of H2O2 in plant physiology and stress responses.
