I haven't done this under anaerobic conditions, but I would explore low temperature, protease inhibitor cocktails and consider gentler membrane lysis such as nonionic detergents or digitonin....

Thank you Shallee for your answer. We have been trying to keep the temperature as low as possible by keep french press in cold room until ready to be used. We also go through only 2 cycles of french press which should be enough to break most of the cells. We tried benzamidine buffer as protease inhibitor but have not shown any promising improvement. We dont add any detergent in lysis buffer, it is only 30mM MOPS.

I'd still go with a cocktail. It costs more, but it's worth covering that base.

And, you mentioned salt...why are you not using standard saline @ 0.15M?

I'd agree with the inhibitor cocktail suggestion, as well as trying different detergents. You could also try a pre-mixed lysis solutions such as TPER depending on your cell source.

To follow anaerobic conditions, u can add a reducing agent (beta-ME or DTT) in all your buffers, to prevent oxidation. add B-ME for Ni-NTA and DTT for GST purifications. also add some salt (150mM NaCl) and some ions (Mg/Mn) to retain protein activity. The main problem seems with your assay buffer. try to work on that aspect.

Thank you Andaleeb for your insights. We perform all of purification steps inside the anaerobic chamber (95% Argon, 5% Hydrogen). Then also should we add DTT or Beta-ME, would not it be harsh for the proteins? My assay buffer is 30 mM MOPS , pH 7.3. Can you suggest any other assay buffers which might help?