The ABI / Apple program has a charting of amount of AA vs. residue
number, which is useful in detecting if your protein is just breaking apart.
Glyco proteins usually run as diffuse bands, so the amount of PVDF goes
up. Try deglycolysating it or concentrating the material before the gel.
As with most protein work, the above are rule of thumb and may not apply to your
particular protein. But it's usually a good starting point.
David T. Chin, Director
UMC Protein Core
e-mail: bcdchin@muccmail.missouri.edu
(573)882-2027 (office and messaging)
http://www.missouri.edu/~pc/
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