Quantification of Glutathione in Caenorhabditis elegans

Samuel W. Caito1, Michael Aschner1

1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.18
DOI:  10.1002/0471140856.tx0618s64
Online Posting Date:  May, 2015
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Glutathione (GSH) is the most abundant intracellular thiol with diverse functions from redox signaling, xenobiotic detoxification, and apoptosis. The quantification of GSH is an important measure for redox capacity and oxidative stress. This protocol quantifies total GSH from Caenorhabditis elegans, an emerging model organism for toxicology studies. GSH is measured using the 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB) cycling method originally created for cell and tissue samples but optimized for whole worm extracts. DTNB reacts with GSH to from a 5′‐thio‐2‐nitrobenzoic acid (TNB) chromophore with maximum absorbance of 412 nm. This method is both rapid and sensitive, making it ideal for studies involving a large number of transgenic nematode strains. © 2015 by John Wiley & Sons, Inc.

Keywords: glutathione; Caenorhabditis elegans

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Caenorhabditis elegans (30,000‐50,000 L1 stage worms or 20,000 L4/adult worms per sample; Caenorhabditis Genetics Center, University of Minnesota).
  • Sterile water
  • Extraction buffer (see recipe)
  • KPE buffer (see recipe)
  • GSH (reduced form) standards (see recipe)
  • DTNB solution (see recipe)
  • GR solution (see recipe)
  • NADPH solution (see recipe)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge
  • Wand sonicator
  • 96‐well microtiter plate
  • Multichannel pipettor
  • Microplate reader (with 412 nm filter)
  • Protein quantification assay kit
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Literature Cited

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