Epitope-Tagging and EM
qiwang at BLUE.WEEG.UIOWA.EDU
Wed Sep 21 09:51:11 EST 1994
Dear netters at methods-and-reagents:
I am seeking your opinions about my proposed epitope tagging
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.
Dept. of Biologcial Sciences
The University of Iowa
e-mail: qiwang at blue.weeg.uiowa.edu
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