Epitope Tagging

Mark Blight blight at igmors.u-psud.fr
Wed Jul 31 05:48:32 EST 1996


Hello,

I am working with an E.coli polytopic membrane protein that is expressed 
at a VERY low level (probably around 100-500 molecules per cell) and I 
have a lot of trouble visualising it on SDS-PAGE. I have a rabbit 
polyclonal antibody raised against a domain of this protein that is 
highly conserved in many homologues. This leads to the detection of 
these homologues in E.coli extracts on Western blots, thereby rendering 
the antibody of little use for immunoprecipitations of specific 
complexes with my protein. It is not yet possible to raise polyclonal 
sera against non-conserved domains.

So, I thought that epitope tagging the protein might help with specific 
detection. I have already His tagged it and used a His tag monoclonal 
antibody but the detection is very poor (probably since its a 
monoclonal). I wonder if anyone out there has had similar experiences 
and/or can suggest other epitope tags - preferably small and with a 
polyclonal antibody raised against it.

Any help would be much appreciated.

Replies by e-mail to : blight at igmors.u-psud.fr

Thanks.
              Mark
____________________________

Dr. Mark A. Blight,
Institut de Génétique et Microbiologie,
Bâtiment 409,
Université de Paris XI,
91405 Orsay cedex,
France.

Tel: +33 1 69416699
Fax: +33 1 69417808
e-mail: blight at igmors.u-psud.fr
____________________________



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