Do you think then that under the anhyrdrous conditions, not even DP is
breaking significantly (in a case where there is low S and T content).
This particular protein, however, is loaded with S and T.
On Wed, 13 May 1998, Steve Latshaw wrote:
> Deb,
> As I recall, a number of years ago George Tarr had studied
> nonspecific background cleavage and had related it to the Ser and Thr
> content of a protein. Under the anhydrous conditions of TFA treatment any
> cleaving of the peptide bond would require a nucleophile and with no water
> around S/T could provide its -OH. He did a modification of the sequencer
> chemistry to include an acetic anhydride treatment after the initial
> coupling step. It may be in one the Techniques in Protein Chemistry as it
> was presented as a poster at one of the Protein Science meetings.
>
> Steve Latshaw
> Chicago Medical School
>
>
> At 01:58 PM 5/12/98 -0700, Deb McMillen wrote:
> >Thanks (Gautam, David, Jacek and Len) for the responses to my question.
> >
> >I have now tried cleaning up the PVDF blot with 50% metoh with TEA. The
> >signal is a lot cleaner, however, it is clear that the protein is still
> >rapidly falling apart. Turns out from the sequence that it has 3 DP
> >bonds. In addition, it has several other bonds that I understand are acid
> >or base labile: 1 GP, 1 DG, 2TP and 2 NG bonds. Does anyone know if any
> >or all of these bonds do break during Edman degradation under the
> >conditions in the ABI 470? Are there any other bonds that are
> >particularly labile under these conditions?
> >
> >Thanks for your help,
> >Deb
> >
> >
> >On Mon, 11 May 1998, Deb McMillen wrote:
> >
> >> Len, hello,
> >> I have a (long) question for you.
> >>
> >> I have a few PVDF samples that have been very darkly stained with
> >> Coomassie Blue. On sequencing, there is one major sequence, but the
> >> background is very high--and gets too high to read the sequence very
> >> rapidly. On one of these samples I know the sequence and will check for
> >> acid/base labile peptide bonds to see if the protein is degrading rapidly
> >> in the sequencer--recently I had a sample with 6 DP bonds and it broke
> >> down quickly and gave a very large "background" which continued to
> >> increase at each cycle.
> >>
> >> I remember though that you had a method for "cleaning up" PVDF blots. Can
> >> it be on these very dark PVDF samples that the Coomassie Blue is hanging
> >> on to cleaved-derivitized amino acids and that these then continue to come
> >> off the PVDF during subsequent cycles, as well as hang on to more freshly-
> >> cleaved amino acids?
> >>
> >> Do some brands of PVDF cause large background in the sequencer?
> >> In both of these current cases I cannot get the client to increase their
> >> protein/PVDF ratio--so there is quite a bit of PVDF in the reaction
> >> chamber.
> >>
> >> And/or do proteins that are glycosylated typically show large background
> >> on sequencing? On proteins that we have thought were glycosylated we
> >> would see cycles with huge peaks at some amino acids (seemed to be a
> >> pattern to the amino acids that were up) and then see fairly clean cycles
> >> scattered in between--supposedly amino acids that were not glycosylated.
> >>
> >> Thanks for any help,
> >> Deb McMillen
> >>
> >> Institute of Molecular Biology
> >> University of Oregon
> >> Eugene OR 97403
> >>
> >
> >
>