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
>>
>
>