You should be able to separate the monomer from dimers and trimers by
reverse phase HPLC.
But then, you lyophilize again and have the same problem--right? You don't
give any
information about the composition, but pH could certainly be a factor in how
fast and whether the aggregation occurs.
SC Howard
-----Original Message-----
From: Thomas Andersen [mailto:AndersT@mail.amc.edu]
Sent: Wednesday, May 31, 2000 10:39 AM
To: Recipients of ABRF List
Subject: peptide size
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 ultracentrifugation? Any other thoughts?
Thanks!
Tom
?------------------------
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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