HA epitope tag problems

Jim Woodgett jwoodget at oci.utoronto.ca
Fri Oct 28 17:07:54 EST 1994


In article <38quqq$88l at mcmail.cis.McMaster.CA>, 
g9013118 at mcmail.cis.mcmaster.ca (K. Legate) writes:
  
> In article <obrien-271094110044 at brass2.med.upenn.edu>, PJOB 
> <obrien at pharm.med.upenn.edu> wrote: 
> >We've been trying to tag a g-protein coupled receptor with the HA 
> >epitope recognized by the 12CA antibody, but have been unable 
> >to immunoprecipitate or Western blot the bugger.  The DNA sequence 
> >looks o.k., but I've heard 
> > 
> I've heard of people having trouble recognizing the epitope if it's 
> tagged onto the c-terminus of a protein. I have it tagging the 
> N-terminus of a membrane protein and have no problems. 

Well, it works the other way around too.  We had a protein that wasn't 
recognized with an N-tag but was fine with a C-tag.  It's empirical.  Out of 
about 20 proteins we've tagged, all have worked with one end or the other, but 
you can't tell which will work before hand.  N-tagging is somewhat easier 
since you can engineer an NcoI site into your ATG and generate a universal 
cloning vector.  The C-tag requires more fiddling.

Jim









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