Western Blot

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Wed Oct 17 07:55:34 EST 2007


Hi Susanne,
i got this link..may be it will solve few of your questions
they say that fixation can be reversible..
kindly share your views
http://www.biotech.ufl.edu/EM/data/formalfix.html

Sudheendra.

On 10/12/07, Susanne Kölare Jeffrey <sjeffrey from u.washington.edu> wrote:
>
> Hi again:
>
> As far as I understand, although I have never done it, using
> formaldehyde fixed tissue (where some of the cross-linking might have
> been reversed) for immunoblotting might require that you use
> antibodies that are specifically made for - or at least, works well
> with - fixed tissue, like the antibodies that are specifically suited
> for immunohistochemistry.
>
> Just one thought - I have a hard time to picture this, pls
> explain...  (I will read your links...) - How can you get an extract/
> lysate if the whole tissue is fixed? How could you separate cell
> organelles, cells and tissues (connective tissue/nerve cells etc.)
> from one another? In other words: Can you really reverse the fixation?
>
> Susanne
>
> On Oct 10, 2007, at 10:10 PM, Sudheendra Rao N R wrote:
>
> > Hi all,
> > thanks for the replies regarding Western Blot using formalin fixed
> > tissues.
> > I searched in protocol online..they say Nitrogen or Argon must be
> > used while
> > extracting proteins from formalin fixed tissues. .but no protocol
> > is given
> > there for that.
> > following is a link to an article in an Elsevier journal (pathology,
> > research and practice) talks about HOPE technique..
> > http://dx.doi.org/10.1016/j.prp.2004.03.009
> >
> > Also, in the following article (PET technique detects PrPSc in
> > paraffin
> > embedded tissues early)
> > http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1868648
> >
> > Also as POW JOSHI forwarded, Calbiochem sells ProteoExtract
> > Formalin fixed
> > tissue kit.
> >
> > @Susanne
> > if antibodies are the limiting factors..is it possible to detect
> > protein(
> > just by using those antibodies which are made by not using synthetic
> > peptides (like using native forms))??
> >
> > Kindly Share your views..
> >
> > Thanks in Advance
> > Sudheendra.
> >
> >
> >
> > On 10/10/07, Sudheendra Rao N R <sudhee26 from gmail.com> wrote:
> >>
> >> Hello all,
> >> can we western blot using protein extract from the formalin fixed
> >> tissues?
> >> Does perfusion of tissue (say brain) has anything to do with
> >> western blot
> >> based protein detection?
> >>
> >> Thanks in advance
> >> Sudheendra.
> >> National Brain Research Centre
> >>
> >>
> >> --
> >> Think before agree
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> >> but STOP thinking
> >> and You Are God
> >
> >
> >
> >
> > --
> > Think before agree
> > Think before you nod
> > but STOP thinking
> > and You Are God
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>


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Think before you nod
but STOP thinking
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