I would raise questions regarding the antibody - possibly post translational
modifications have masked the epitope and thus the contaminating peptides
are not contaminants but rather the answer.
----- Original Message -----
From: Katheryn Resing <Katheryn.Resing@Colorado.EDU>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Sent: Saturday, March 04, 2000 4:08 AM
Subject: Is this a protein?
> 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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