My boss says that for as long as he can remember it has been known that EDTA does not go through dialysis membranes very well, but I can't find any documentation or mention of it anywhere. Does anyone have any references or anything to document this?

Hi, you can check any protein purification protocol book and i hope you'll get the answer

No - none of the books, protocols, manufacturers that I have seen mention it, but we have experience that this is so. Has no one ever documented it?

There are some examples of what you mention in the literature. Check this:

Escherichia coli nucleoprotein has been found to undergo thermal denaturation approx. 5 degrees C lower than purified E. coli DNA, measured in 0.25 mM sodium EDTA, pH 8.0 (Searcy, D.G. (1975) Biochim. Biophys. Acta 395, 535-547). This is now shown to be an artifact due to the impermeability of dialysis membranes to sodium EDTA, especially at low concentrations such as 1 mM or less.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=812553

To remove EDTA, the two samples were extensively dialyzed against ultra-pure water (Milli-Q), at 4 °C, and the EDTA-SM was freeze-dried (...) The presence of residual EDTA after intensive dialysis of EDTA-SM was also observed in amino acid composition determination as EDTA eluted near to the histidine, precluding from detecting this amino acid.

http://www.blackwell-synergy.com/links/doi/10.1046/j.1432-1033.2002.03203.x/full/

Thanks! I had found the first reference, but didn't have the second one. I appreciate you taking the time to find it.

Does this apply to EDTA-metal complexes (e.g. with Mg or Ca) or just free EDTA?

Yes - in the papers referenced above - one has Ca the other uses Na EDTA. It seems that you have to do very extensive dialysis to get the EDTA out or in.

So, it is not easy to get rid of EDTA by dialysis. Does gel filtration work any better?

I don't have any direct experience, but since the mechanism of separation is different I would think that gel filtration would work. Maybe someone else has data on this?

In the diccussion above, no one suggested a reason for EDTA's resistance to passing through the membrane. Without knowing this it is hard to evaluate whether or not the same thing would happen with gel filtration.

I imagined that maybe each metal ion is chelated with 'arms' from a few different EDTA molecules. If this is true the EDTA and ions could form a large network that the dialysis membrane would see as a very big molecule. If so, you might get the same effect with gel filtration.....but I'm not a chemist, so I don't really know if this all makes sense.

I have worked with a peptide sensitive to metal catalyzed cleavage, so the presence of high levels of EDTA was very protective, but unfortunately difficult to remove downstream by dialysis. One strategy that has been helpful is to include a separate dialysis bag in the dialysis buffer containing Chelex from Bio-Rad or Sigma which is basically immobilized EDTA. The Chelex can be kept separate from the second dialysis bag which contained the peptide.

Follow up: I tried getting rid of the EDTA in my protein solution by TCA precipitation of the protein. That was pretty stupid because TCA lowers the pH of the solution and preciptates the EDTA also. So I redissovled and tried getting rid of it by gel filtration. Much better results! And only a fraction of the work. Now I know...