I have been trying to purify a 76 kDa protein and it was a hard process. I tried His tag but I didn´t get good results but when I used it with MBP I had a very good result and big bands after using the amylose resin but after that when doing a size exclusion chromatography I notice that my protein is aggregating. Any ideas how to solve protein aggregation?
Note: I use large scale autoinduction cultures and temperature of 16 degrees o/n
Buffer used : 30 mM tris 7.5, 200 mM NaCl, 1mM DTT, 1mM EDTA + 20 mM Maltose for elution
In my experience you should try dialyzing your protein against different buffers. Your protein could be unstable in the buffer your are using bat stable in other buffers. I have detected many times different behavior depending salts concentration. I usually try high salt concentration buffers, medium salt concentration buffers and low salt concentration buffers and empirically check which one are the best.
I suggest you some buffers that I usually try.
20mM Tris + 500mM NaCl ( High salt concentration)
NaCO3H 333mM ( medium salt concentration)
PBS + 10% glycerol ( medium salt concentration)
20mM Tris + 5% Dextrose ( low salt concentration)
Thank you all, first this protein is a part of the nuclear export protein chain and no i didn´t cleave the MBP before (i suppose after cleaving it would be much worse). The IP of this protein is around 9.5 and the buffer am using has a pH of 7.5.
In addition to Ugutz's suggestions, make sure you add 10-15% glycerol in all your buffers. It serves to atabilize the protein. You may have to reduce the amount to 5% when running chromatography columns, to prevent high back pressure. Do you know that your protein exists only as a monomer? Is is possible that it oligomerizes? In case it oligomerizes, you may not be able to see that in an SDS PAGE gel, but it would run slower in a gel filtration column.
Maybe you should use a buffer more closer to the pI of your protein? Like Carbonate buffer?
All the best with your purification.
Thank you all for your suggestions , i will take that into consideration.
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