cpatil at itsa.ucsf.edu
Tue Feb 4 13:43:26 EST 1997
In article <199702032102.QAA19223 at pilot19.cl.msu.edu>,
Ivan J Delgado <delgadoi at PILOT.MSU.EDU> wrote:
> A question regarding glycosylation of proteins and how that looks in a
>western. I have two different antibodies raised against the same protein. When
>I use these on a western, in one case I get a tight band while (antiboby 1) and
>in the other I get a smear of smaller molecular weight bands (antibody 2). I
>don't think it is due to degradation since the first antibody gives a clean
>signal (protein sample was the same for both and the same wetsern). Could this
>be glycosylation?. How could one test for it?
It's almost definitely not glycosylation; glycosylation _adds_ molecular
weight to a protein. Protein glycosylation is used by those in the
translocation field as evidence that a protein has been translocated into
the ER or Golgi; the glycosylated form shows up as a single clean band on
the gel at higher molecular weight.
What about the possibility that Ab#1 recognizes a less stable part of the
protein than Ab#2, so that after partial proteolysis/degradation, the
epitope for Ab#1 is gone (so you only see the undegraded full-length form)
whereas the epitope for Ab#2 is still present (and you therefore see the
degradation ladder)? A prediction of this theory is that the antibodies
will recognizes different tryptic fragments of the purified protein.
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