anti-HA immunofluorescence

bram at mbcf.stjude.org bram at mbcf.stjude.org
Tue Feb 14 16:49:19 EST 1995


In article <3hav6j$c7j at charm.magnus.acs.ohio-state.edu>, tchang at magnus.acs.ohio-state.edu (Tien-Hsien Chang) writes:
>      We are trying to localize a HA-tagged protein in cerevisiae by using BMB's
> anti-mouse monoclonal.  We would appreciate that someone who has successfully 
> done the same or similar (using anti-HA) thing before to provide protocols or 
> suggestions.  I heard that this antibody appears to recognize nonspecific 
> proteins (containing HA-like epitopes) in the cerevisiae.  If so, how can this 
> be solved?  We are thinking about doing a preadsorption to yeast powder.  Is 
> this a good way?  Finally, does the strain genetic background matter?
> 
>      Thanks in advance.
> 
>                          Tien-Hsien Chang

Just my two cents on the 12CA5 HA monoclonal. I think it is useful to 
titrate (down, way down) the concentration to maximize specificity. This
holds for immunofluorescence and for Westerns. For instance, your last
post referred to the recommended concentration of 10 mcg/ml, but I have
seen better results with concentrations as low as 1 mcg/ml.

Also, it seems to me unlikely that you can preadsorb a monoclonal antibody
to improve the specificity, since there are no "non-specific" antibodies
in there to remove!

Good luck!!
rick



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