its been a while since my last post
I'm currently struggling with a problem i can't get a hold on.
We are trying to purify a Fab, that we express in the periplasm of E. coli, using Lambda FabSelect column from GE.
The protein should normally elute with 100 mM acetate buffer with pH 3.
We are not able to elute our protein at pH3 but it can be eluted at pH2 if we lower the pH of our elution bufffer. The protein than elutes perfect and looks rather nice on SDS-PAGE (we have good concentration and purity, the protein looks the same as our standard Fab in a non-reducing and reducing SDS-PAGE).
At that point everything seems to be fine.
But now the real trouble starts.
The protein that we have now in 100 mM acetate buffer at pH2 can not be shifted to e.g. pH 7.4 (PBS) without precipitation (it seems that it precipitates around its pI).
We tried to do buffer exchange using different methods, eg. by using PD-10 columns ...her it migrates again with all the salt and elutes lately (so i assume it precipitates on the column and then gets dissovled again).
My problem currently is that i have no clue why it takes pH2 to elute from the affinity column ...and why it then it becomes so resistant to buffer exchange.
Do you have any suggestion or hints how to solve this issue?
Many thanks in advance and all the best for the new year,