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
>