Epitope-Tagging and EM

[wang qi]

Dear netters at methods-and-reagents:
  
   I am seeking your opinions about my proposed epitope tagging 
experiment.

   I am working on a plant protein which has a very controversial 
localization. The polyclonal antiserum we raised in a rabbit did not give 
us any useful information at all. We then went for monoclonal antibodies 
and one monoclonal antibody showed some kind of tissue-specific pattern 
using immuno-fluorescence microscopy. The same monoclonal antibody, 
however, could not identify any antigen at the electron microscope level. 
To further test the tissue-specific binding shown by the monoclonal 
antibody and to localize this protein at the EM level, I thought epitope 
tagging might be a powerful tool. Although the technique is a relatively 
straight-forward one in the published papers, I do feel that I need to 
ask a few questions before I can go ahead with the experiment:
1). Which epitope is a better choice in terms of EM localization?
2). Should I use duplicated or triplicated epitopes?
3). Where should I insert the epitope(s) in the protein? N-terminal? 
C-terminal or in somewhere between? 

    Answers to these specific questions as well as any general 
suggestions will be greatly appreciated? I prefer that you direct your 
responses to me and I will summarize them and post it back to the group if 
there is a need. Thank you all very much.

QiWang
Dept. of Biologcial Sciences
The University of Iowa
e-mail: qiwang at blue.weeg.uiowa.edu