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More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.

 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.