Tom,
I would think you would want to consider how you want to be able to
handle the peptide after purification, also. If you use RP HPLC with a
TFA based chromatography, you will probably need to lyophilize off the TFA
before you can use your peptide--leading to more dimerization.
I wonder about an ion exchange chromatography--like a SCX column like the
poly sulfoethyl aspartamide column that I use to separate charged species
(see J Chromatography 594:75 (1992) for an example of an application of
this column) or its compliment, an SAX column
(these are from Poly LC,, but I purchased mine from the Nest
Group--you might be able to purify your peptide under conditions that the
eluant ends up being a solvent that prevents dimerization of the peptide
on storage. You can use organic solvents on these columns too if you need
to disrupt hydrophobic interactions in addition to salt interactions (or
enhance one or the other of these interactions). In addition ion exchange
columns have a much larger capacity for material than gel filtration
columns.
If you want more ideas on this, the technical support at both the Nest
Group and Poly LC have proved to be very helpful on separation questions.
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403
On Wed, 31 May 2000, Thomas Andersen wrote:
> Hey ABRFers-
>
> I need some help.
>
> Do you believe that I should be able to separate an octapeptide from its dimer? And if so, by what technique?
>
> I have a well-characterized 8-mer that is just the niftiest little peptide you'd ever want to see, but upon storage in the lyophilized state, it loses activity. We have shown that there is no chemical modification of the peptide, and if we treat the stored peptide with urea, we can recover activity. Therefore, we think the peptide aggregates in storage. Also, we have shown that an earlier, larger version of this peptide, a 34-mer, does indeed aggregate in a very complex manner during storage in the lyophilized state. The 8-mer does not aggregate to a huge complex, so I think it is a dimer, maybe a trimer at largest. But I can see no resolution on high pressure gel filtration chromatography. I believe that I will never see much resolution, even if I hink around with pH, buffers, temperature, whatever. But I'd sure hate to be wrong about that. Do you think it is worthwhile trying harder to separate 8-mer from 16-mer? HPLC? Low pressure gel filtration? analytical ul!
tr!
> acentrifugation? Any other thoughts?
>
> Thanks!
> Tom
>
>
>
> Ø------------------------
> Thomas T. Andersen, Ph.D.
> Director, Protein Chemistry Core Facility
> Albany Medical College
> Albany, NY 12208
>
> 518 262-5368
> anderst@mail.amc.edu
>
>
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