[Protein-analysis] Re: Ammonium Bicarbonate in IE Chromatography

Bruce D. Ray bray at iupui.edu
Fri Oct 6 17:26:03 EST 2006


Apparently this did not propogate from the news server to which I
posted it
originally.

In article <1157020009.287991.209120 at b28g2000cwb.googlegroups.com>,
pawel.stocki at ncl.ac.uk wrote:

> Does anyone have some experience in using ammonium bicarbonate buffer
> for gradient elution from Q-Sepharose or other IE? I would like to use
> it instead of NaCl gradient. The sample will be subjected for further
> MS analysis that is why I would like to use volatile salt. I'll
> appreciate any comments.

I've used ammonium carbonate/bicarbonate, pH 8 and 4 C for
purification of ATP/ADP/AMP mixtures both with QAE and with
DEAE media to prepare isotopically labeled mononucleotides
at high purity.  Major considerations are:

   1. Ammonium carbonate/bicarbonate, pH 8 must be fresh when used.
      This is a volatile buffer and will decompose over several days
      even at 4 C.
   2. Don't go above 1 M bicarbonate.
   3. Equilibrate the column with the bicarbonate buffer.  As long as
      it stays at pressure after equilibration, voids will not form.
   4. After chromatography, re-equilibrate the column with a
non-volatile
      buffer.  This is a precaution to prevent void formation.  I've
      never had a problem with void formation, but this buffer could
      form gas bubbles under some circumstances.
   5. Rotary evaporation with methanolysis to decompose residual
      ammonium bicarbonate works better than lyophilization (unless
      the lyophilizer pump is protected with an ammonium trap) because
      the ammonia tends to accumulate in the vacuum pump oil of the
      lyophilizer.  Typical rotary evaporation setups use either a
      water vac or a diaphragm pump and these do not accumulate
      ammonia.
   6. Do not leave collected fractions of interest standing for
      long periods after chromatography.

I hope this is helpful even after such a long time.



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