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HA Immunoppt

Maria C. Costanzo mcc9 at cornell.edu
Wed Oct 16 11:57:11 EST 1996


In article <forte-1110961601490001 at 137.53.73.42>, forte at ohsu.edu (Michael
Forte) wrote:

> Hi Folks,
> 
> We are trying to immpt. a yeast mitochondrial membrane protein which has
> been tagged with the HA epitope.  In our hands, the only way we can access
> the epitope is to spheroplast, solubilize in 1% SDS and dilute the
> extracts to 0.1% SDS before adding antibody.  As far as antibodies, we
> seem to get better recovery if we use the HA.11 monoclonal from BabCo
> rather than the 12CA5 monoclonal.  The trouble with this is that we are
> getting background that we are having trouble eliminating.  Has anyone
> tried the BabCo polyclonal antibody to the HA epitope for immpt. and is
> the background better?  Any suggestions or experience here will be
> appreciated.
> 
> Mike Forte
> 
> -- 
> Mike Forte
> forte at ohsu.edu or
> forte at mtwo.com

We have been trying to immunoprecipitate a mitochondrial ribosomal protein
using the 12CA5 monoclonal.  We found that adding some goat anti-mouse
polyvalent Igs to the immunoprecipitation before the protein A helps
somewhat.  I suspect that part of our problem, like yours, has to do with
the accessibility of the epitope. I seem to recall hearing, but can't
remember from whom, that the polyclonal anti-HA does not work well.

Hope this helps -

Maria
Maria



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