antibodies to histag proteins

Jeff "Newt" Sekelsky fznewt at peseta.ucdavis.edu
Mon Sep 16 10:29:06 EST 1996



On Sat, 14 Sep 1996, David Micklem wrote:

> In article <3239CC2B.3D41 at bimcore.emory.edu>, TJ Murphy
> <medtjm at bimcore.emory.edu> wrote:
> 
> >Malini Rajagopalan wrote:
> >> 
> >> Hi,
> >> 
> >> I am planning to make antibodies to histag proteins without cleaving
> >> the histidine tag. However, I am concerned about getting more
> >> background in western blots and immunoprecipitations using antibodies
> >> made to uncleaved histag proteins. Does anyone have any experience in
> >> this matter ?
> >> 
> >> Malini Madiraju
> >
> >The antibodies will work well IF used to detect overexpressed protein
> >made in bacteria.  If you plan to express the protein in eukaryotic
> >cells, the antibody will not work well unless it is a massive
> >overexpression system.  They are just not sensitive enough for that.
> 
> Huh? Why not?  Surely the antibodies will be just as high affinity as
> those prepared against non-His-tagged proteins.
> 

I think the author of the first response misinterpreted the question - it
looks like a comment on the antibodies to the 6xHis tag itself, for
example the antibodies produced by Qiagen (and others) that recognize the
epitope MGRSHHHHHH.  I haven't used them, but previous posts to this
newsgroup indicate that they are only useful in cases of very high
expression.

As for the original question, I've used the 6xHis tag for two different
proteins.  For the first, I got good antibodies specific to the protein I
wanted (on Westerns, at least), so background isn't necessarily going to
be a problem.  If it is, you should be able to preadsorb against bacteria
expressing a control 6xHis tagged protein to remove any antibodies to the
tag.  For my second protein, I seem to have gotten reasonable antibodies
to the tag itself, since they recognize the tagged first protein just was
well as the tagged second protein.


Jeff Sekelsky





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