dialysis membranes and EDTA

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

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

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
Quote:

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/

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

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

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...

Hey, have you gotten a good way to seperate EDTA from protein?

Hi Snygg !!

At what scale you whant to seprate it
If it is small scale centricon is good option!!!

Rajeshwari

Hey,Rajeshwari,
 How small will be suitable for centricon ?
 I run my protein through heavy-metal column, such as Pb-column. Then used EDTA to elute. My protein is with EDTA and metal ion. Can certricon get rid of both Pb and EDTA?