dialysis membranes and EDTA

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SueC.
SueC.'s picture
dialysis membranes and EDTA

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?

Rajiv Tyagi
Rajiv Tyagi's picture
Hi, you can check any protein

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

SueC.
SueC.'s picture
No - none of the books,

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?

badcell
badcell's picture
There are some examples of

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/

SueC.
SueC.'s picture
Thanks! I had found the

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

Paul Weinreb
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Does this apply to EDTA-metal

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

SueC.
SueC.'s picture
Yes - in the papers

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.

ike9898
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So, it is not easy to get rid

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

SueC.
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I don't have any direct

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?

ike9898
ike9898's picture
In the diccussion above, no

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.

TigerShark
TigerShark's picture
I have worked with a peptide

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.

ike9898
ike9898's picture
Follow up: I tried getting

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...

Snygg
Snygg's picture
Hey, have you gotten a good

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

Rajeshwari patel
Rajeshwari patel's picture
Hi Snygg !!

Hi Snygg !!

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

Rajeshwari

Snygg
Snygg's picture
Hey,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?