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Fab antibody fragment purification


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#1 pDNA

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Posted 13 January 2017 - 01:33 AM

Dear people,

 

   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,

pDNA



#2 mdfenko

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Posted 13 January 2017 - 05:33 AM

a couple of things;

 

you should be able to resuspend the precipitated protein after the pH is shifted away from the pI, unless the protein is actually denatured rather than isoelectrically precipitated.

 

you can avoid the pH adjustment step by the pre-addition of some 1M tris, pH 9 to the collection tube(s). after elution, you may have to mix them to ensure that the protein is suspended.

 

you can obtain a free handbook on antibody purification (and others) at this ge lifesciences webpage.


talent does what it can
genius does what it must
i do what i get paid to do

#3 Missle

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Posted 18 January 2017 - 12:09 PM

I second the addition of some 1M tris to the collection tubes.  You could also try buffer exchanging into a different buffer.  If it crashes out at 7.4, try putting it into a pH 5 or 6 buffer.  Good luck!






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