I use the Ellmann method for the determination of glutathione. There are so many options as a buffer. I prepared a 0.1 molar 8.4 pH Tris buffer for the reaction mixture. I have a trismza base (sigma aldrich: https://www.sigmaaldrich.com/TR/en/search/77-86-1?focus=products&page=1&perpage=30&sort=relevance&term=77-86-1&type=cas_number) . I made a pH adjustment with NaOH and HCl. I prepared the Ellmann reagent in the same way on a pH 7 buffer. According to my readings, sodium phosphate buffer or tris HCl-EDTA mixture is also used. Can someone who has worked on this issue before tell me which tampon I should use and how to prepare it?
Sulfhydryl quantitation using Ellman's reagent is not very sensitive to which buffer is used, as long as the pH is neutral or basic. Mildly basic Tris and phosphate should both work fine. The only observation I will make is that the stability of the absorbance signal may be affected by the choice of buffer. A long time ago I found that the color faded slowly in an organic buffer (I think it was MOPS) and was more stable in phosphate, but this would only matter if you need the absorbance signal to be stable for a long time, instead of reading it right away.
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