Currently, I am purifying a protein which has a fairly good concentration after elution. However, after size exclusion chromatogrphy, protein is aggregated. What can be the possible solution?
Hi there,
What is the elution buffer composition? Is your protein stable before SEC when in the same buffer? It might be the dilution effect of SEC is enough to provoke aggregation? Or the protein is actually losing something essential to its stability during SEC (cofactor, ligand, bound ion...). If it's the case you would consider adding it in to the elution buffer.
Hope it helps.
The simplest suggestion is to add a reducing agent to the elution buffer and try again. You have also to determine whether it is aggregation, or self-association. In this case a suggestion is to dilute the protein during the chromatography.
Thanks a lot for the reply. I am going to add 2-mercaptoethanol with my elution buffer. My target protein is a His-tagged protein. Therefore, elution buffer contains 300mM NaCl, 300mM Imidazole and TRIS PH 6.0.
If there is significant leaching of the metal ion during IMAC, His-tagged proteins may aggregate by forming complexes with it (see e.g. PMID 9668967 and 16904956). Try adding EDTA and checking what happens to your SEC profile.
I tried SEC separately by keeping His-tag and removing His-tag. However, in both cases aggregation of protein took place. The composition of my SEC buffer is 20mM Tris HCl PH 8, 300mM NaCl.
Hi Mohammad, can you give some more details about where you are getting your protein from?
E. coli exclusion bodies? Mammalian tissue culture medium? Cell extracts? Yeast media or extracts? Insect cultures?
I have used E. coli Bl21 (DE3) strain for the production of my protein.
Hi Mohammad, is your protein isolated from inclusion bodies? Does it have free thiols?
No it was not isolated from inclusion bodies. Regarding thiol, it has 4 cysteine on it. However , its structure is not determined yet. Therefore, I am not sure those cysteines are in outer portion or not.
Hi Mohammad, do you know if the cysteine thiols are free in your expressed protein?
If so, then that might be a factor in causing protein aggregation after SEC. To test this, you could alkylate your prep using iodoacetamide or maleimide and see if the protein aggregates after SEC.
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