Western blot advice needed

D.K. dk at no.email.thankstospam.net
Wed Oct 23 22:34:15 EST 2002


Mike Levin <mlevin77 at attbi.com> wrote:
>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!!

It's a gel, not Western that needs to be dissociated. Similar 
things happen with a number of membrane proteins. Some remedies 
that worked in other cases: 

Do not heat at all - leave sample at room temperature,
use lauryl sulfate in place of SDS, use sucrose in loading buffer in 
place of glycerol, use 200 mM DTT in loading buffer in place of bME,
increase detergent concentration in loading buffer 2X. 

Additionally, if the source of the problem is indeed lipid, perhaps 
extracting your samples with chloroform first might help.

DK



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