L. M. Weaver
lmweaver at students.wisc.edu
Fri Apr 12 11:11:38 EST 1996
Some time ago I posted a request for info on epitope-tagging in
Arabidopsis, specifically for which epitopes are best to use. The
responses I received, and what I gleaned talking to people, can be
summarized as follows:
1. There is much rumor and work-in-progress involving immunodetection
using antibodies to HA1, His Tag, and T7 Tag, but I haven't talked to
anyone who has them up and running, or who has compared them with each
other or Myc.
2. The clear winner in the epitope popularity contest is Myc, detected
with the Oncogene 9E-10 monoclonal antibody. A lot of people are using
this, a lot of those who responded are getting it to work, and opinions
over what makes for a good construct tend to differ.
3. The c-Myc epitope comes in two flavors (that I know of). One has a
"minimal" sequence of EQKLISEEDL, and the other tacks on some extra
residues to give EQKLISEEDLLEK. Some people claim the latter is better,
while plenty of others have had great success with the former.
4. Myc tag constructs also come in two flavors -- those with only one
epitope, and those with several repeats (six is a common number). Again,
some people will tell you that 1 is all you need, while others say you
really want it repeated.
5. Reports of background differ greatly. I originally posted the question
because someone in my lab was using Myc Ab to detect a quite non-abundant
tagged protein, and having lousy luck doing it. He detected it all right,
but assorted background bands were very much stronger than the signal,
which made his life more difficult than it would otherwise have been.
Nobody else I spoke with had that problem, but then nobody else works with
the Demon Protein from Hell, and so presumably does such long exposures.
The background bands he saw were very reproducible, but differed from
tissue type to tissue type.
The only summary of all this I can come up with is that Myc works well
except for when it doesn't, which is to say when you have a very rare
protein which runs at the same size as a background band in the
particular tissue you are working with. If possible it is probably best to
use a tag containing multiple repeats of the 13 residue epitope -- less
will probably work, but if you can scare up an appropriate construct why
not use it all? I don't know how multiple repeats will affect the
likelihood of the tagged protein being functional, but for my purposes it
doesn't matter (I don't have a knockout, you see, but that's a different
story...). Epitope tagging still seems basically to mean Myc tagging,
which often but not always works. Opinions on why that is the case differ
(and few side-by-side comparisons of the different constructs have been
done). The only thing to do is try it and see. Same story, different
U.W. -- Madison
PS Any responses are still welcome, either mailed to me or posted to the
newsgroup. This is clearly a topic of interest to a considerable number of
people (I got more responses from people who wanted to know what I heard
than from people who had done the experiments).
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