For gel exclusion chromatography it is generally adsvisable to include 100 mM NaCl or equivalent ionic strength in the elution buffer to suppress nonspecific adsorption. In addition the pH and ionic strength of the elution buffer should be compatible with your protein folding stability. It is possible that your protein requires significant ionic strength to remain soluble or properly folded. It seems likely you lost protein in GEC due to adsorption or precipitation. Try adjusting the pH and ionic strength of the elution/dialysis buffer appropriately. It is unlikely you are getting proteolysis in a highly purified protein sample.
Cheers, Roger Rowlett Professor Dept. of Chemistry Colgate University On 5/18/10, intekhab alam <faisal...@gmail.com> wrote: > Hello > I am trying to complex two proteins of 57 and 18 kda and using pET 22b as an > expression system. I purified both with His tag and the purity is quite nice > after Ni-NTA (Buffer is Tris,Nacl and Imidazole). After purification with > Ni_NTA i tried to improve my protein purity using gel > filteration(superdex-200). I successfully got the 57 Kda with a high purity > (Buffer is Tris and nacl) but with the smaller protein i didnt get anything > after the column. when i tried dialysis to remove imidazole from this 18kda > protein it degraded.i tried protease inhibitors as well but still the > protein is not stabilised. Can anyone suggest me something to get out from > this. I am stuck at this stage. suggestion are highly welcomed and > appreciated. > > -- > INTEKHAB ALAM > LABORATORY OF STRUCTURAL BIOINFORMATICS > KOREA UNIVERSITY, SEOUL > -- Sent from my mobile device
