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I have a very peculiar experience with an antibody against a membranebound
receptor. Using the microwave-citrate procedure on parafin embedded tissue
I get clear and specific labelling in the exact location, where it is known
to exist from functional studies. However on westerns it only recognizes
the immunizing peptide...and on cells transfected with the protein
(functional test show they express it) I get no labelling.

Regarding the westerns I have some ideas about why I can't see it, but the
cells really irritating.

The urea procedure sounds interesting, and I would like to try it to see if
I can get the antibody to work using this procedure. Could you give some
further details or references? Did he simply wash the cells once in 6M urea?

regards

Sebastian Frische
Dept og Anatomy, University of Aarhus, Denmark

At 18:40 16-05-2003 -0400, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>         The first thing I want to know is what type of protein your antibody
>was raised against.  Is it native or denatured or is it one of the
>bacterially produced peptide sequence?  Also are you working in the same or
>cross species?  A majority of antibodies raise against denatured protein
>species will not react against native proteins as fixation does not
>necessarily denature the protein, particularly if it is a monoclonal and not
>polyclonal.  Yes it will work in a western blot where you are working with
>denatured proteins.  The question is whether it will immuno-percipitate,
>where you have native protein.  The binding site of the antibody may have
>been uncovered during its denaturing process.  You would then have to treat
>your material in the same manner.  A while back I had a student working with
>a protein that was denatured with 6m urea and his antibody raised against
>that.  He could not get any immunolabeling until I told him the treat his
>cells with urea.  The cells then lit up like a Christmas tree.
>
>Joe Goodhouse
>Confocal / EM Core Laboratory
>Department of Molecular Biology
>Princeton University
>609-258-5432
>
>Visit us at  http://www.molbio.princeton.edu/facility/confocal/index.html