Western blot advice needed

Mike Levin mlevin77 at attbi.com
Wed Oct 23 17:15:47 EST 2002


Hi all -

   We are trying to run a Western blot on a protein in the lysate from frog
embryos. We have a really nice antibody to the protein we're interested in.
This thing works great in immunohistochemistry and a competition assay with
the peptide it was generated against is very clean, suggesting that the
binding is quite specific. Here's the problem. This target is a 16 kDa
proteolipid subunit which forms hexamers in vivo. On the Western, we see
bands corresponding to monomers, dimers, trimers, etc. all the way up to the
predicted hexamers. This happens even though our blot contains SDS
(beta-mercaptoethanol, etc.) - in theory, it should be dissociated and all
we should see is the monomer. For some reason, this thing is really
difficult to dissociate. Heating the sample just makes it worse (possibly
because of the lipid portion). Does anyone out there have any ideas as to
how to modify a basic Western blot protocol to make sure that such complexes
are dissociated? If you have any thoughts, please email me at
mlevin at forsyth.org. Thanks in advance!!

Mike Levin




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