Best ways to tag a epitope?

Namjin Chung nc1 at acpub.duke.edu
Wed Sep 11 15:59:34 EST 1996


Dear Netters,
I am trying to tag either hemagglutinin or myc epitope to the C-terminal 
of my-favorite-gene (MFG), so that I could easily isolate the protein Mfg 
with antibody.

I have read a paper where the author tagged three tandem repeats of HA to 
immediately downstream of C-terminus, and she got a clear band when 
immunoblotted.

My questions are:

1.  Is it better to have any number of tandem repeats than to have just 
single copy of an epitope when I tag my MFG?  If so, what would be a good 
method to tag multiple copies?  I wondered PCR or in vitro mutagenesis, 
but PCR with about 100-base primer seemed unrealistic, and for in vitro 
mutagenesis, insertions are (as my knowledge goes) good for the maximum 
number of around 30 bases.

2. When I tag MFG, is it alright to tag to the immdediate downstream of 
the C-terminus?  Shouldn't there be a spacer peptide sequence?

I would appreciate any advice for this.  



************************************************************************
* Namjin Chung                             *                           *
* Program in Molecular Cancer Biology      * Email: nc1 at acpub.duke.edu *
* Duke University Medical Center, Box 3345 * Voice: +1 (919) 684-2363  *
* Durham, NC 27710                         * Fax  : +1 (919) 681-8253  *
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