Good luck,
Steve Latshaw
Chicago Medical School
At 11:38 AM 5/13/98 -0700, Deb McMillen wrote:
>Steve, Thanks for the acetic anhydride idea. I will look that up.
>
>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
>> >>
>> >
>> >
>>
>
>