Could anyone advise me how can I improve or change my purification protocol to avoid aggregation of my protein?
My protein is about 25KDa (it consists of Thioredoxin, His tags and 60AMK, mean incorporated in pET32B).
I am using Rozetta Cells and cultivate them on temperature 18degree after induction (25degree) of 0.5mM IPTG.
After purification step (chelating sepharose) in PBS Buffer, I leave sample in dialysis overnight and after I do Gel filtration on Superdex 75.
before Superdex I concetrate the sample (there is no precipitation in sample). But on Superdex my protein runs like 200KDa.
On SDS gel it is only my 25kD band.
Please could you help me ? any suggestion?
Thanks a lot