Here's a puzzler for you all. We have been trying to get peptide sequence
from an in-gel digest of an SDS-PAGE band that runs as a relatively sharp
band at 18K (these are from a relatively clean membrane preparation that
has only a few bands). There is another protein at 15 K which is known and
we get some contaminating peptides from the 15 K protein in the 18K band.
The 18K band does not react to an antibody to the 15K band, so the amount
of the 15K contaminant has to be a relatively minor. BUT we have been
unable to identify any other peptides by maldi, lc/ms on Poros or Vydak C18
(although we see the 18K peptides in nice intensity every time), either
using trypsin or chymotrypsin as digesting enzymes (in-gel).
We have tried rerunning the two bands on a second gel, and the 15K came out
of the first gel piece almost quantitatively, but there was no trace of the
18K in the second gel, and it appeared to still be in the original gel
piece because we could restain it.
The 18k does not seem to be retained by the PVDF paper; after transfer
and fast greeen staining we cannot see the 18kd (but we can see a strong
15kd band). The 18kd does seem to be retained somewhat by nitrocellulose
paper as visualized by fast green. We tried digesting it after
electroeluting it onto nitrocellulose and only got a trace amount of the 15
kDa contaminanting peptides.
We wonder if it might be something else, other than a protein, for example
a complex glyolipid.
Thanks in advance for any help with this puzzle.
Katheryn Resing
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