Buffer exchange on unfolded proteins

[jdashcup]

I am trying to get my protein into a deuterated buffer for NMR. I tried using the Millipore centrifugal filtration units and lost most of my sample to it. I am thinking the protein is sticking to the membrane. I am looking for a substitute buffer exchange method. Dialysis isn't an option because of the cost and quantity of solvents required. Any suggestions would be helpful.

 

Thanks.

CUP

[CharonY]

You could try other filter materials for ultrafiltration (I assume you sued the amicon columns and you used the correct MWCO?).

 

Otherwise you could make a cleanup using affinity columns (often sold as desalting columns). However I am not sure whether they are really much cheaper than a small dialysis tube. In fact, I think they are roughly in the same price range. Why is the quantity of solvents limiting?

 

Or you could precipitate the protein and wash the pellet to remove buffer and salts (though some proteins do not easily resolubilize).

[Adam Green]

The Millipore Amicons just use a Regenerated Cellulose based membrane, you could try the Sartorius PES based membrane filters. You could the further passivate the ultrafilter by washing with a buffer like Tween, Triton, PEGS to bind to the non-specific binding points in the membrane. 

[BabcockHall]

Some filtration units are optimized for speed, as opposed to recovery.  Microdialysis might be possible.