I have been trying to elute a GST tagged protein from Glutathione 4B Sepharose using reduced glutathione. The reduced glutathione 250 g bottle is from Sigma and was opened in 2016. It does not elute my protein, even at 100 mM. I think that opening and closing the bottle for two years has gradually oxidised the peptide and decreased it's ability to displace the GST from the beads.

I have tried using 100 mM Glutathione with 80 mM DTT to elute the protein, but it failed. I have now tried 100 mM Glutathione with 150 mM DTT and 100 mM Glutathione with 150 mM beta mercaptoethanol (two parallel experiments). I am yet to run the elutions on a gel but I have a suspicion it won't work.

Has anyone tried doing this? Is there a optimal reducing agent concentration? When the bottle was freshly opened proteins eluted with only 10 mM Glutathione. It is an expensive chemical to buy new so I am trying to see if it will work and at what conditions.