Hello.

I am new to the GSH assay and I am learning about it online. I performed it this week for the first time.

My protocol said to add DTNB and GR to my GSH standards + samples - allow 30 sec the conversion of GSSG to GSH - and THEN - add NADPH and measure absorbance.

Is not NADPH essential for the conversion of GSSG to GSH? Why adding it later?

Also. Can I add just DTNB and measure the absorbance (initial GSH) and then add GR+NADPH and measure absorbance again to measure the total GSH (Initial GSH + reduced GSSG)?

So I can calculate also GSSG (GSHt - GSHi) with this assay (without using vinylipyridine) ?

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