FLAG epitope replies

S. Kjemtrup skjemtru at email.unc.edu
Fri May 23 10:42:56 EST 1997


Dear Netters!
The following are the e-mail messages that I received regarding the FLAG
epitope question that I posted a while ago. Thanks to everyone who
e-mailed replies.
Susanne Kjemtrup


I have used the flag tag at both the N- and C- terminus of bacterial
proteins. My signals are best with C-terminal tags, but I'm sure it will be
protein dependent. Westerns on bug extracts are pretty clean, one or two
contaminating bands, depending on the bug and on the exposure time.
Alkaline phosphatase reactions are very clean, ECL system gives more
background... but overall, it is less than myc Ab background. Expression of
bacterial tags in arabidopsis and tobacco are similarly clean. I have not
tried to express these proteins in plants, but the Grussiem lab has and
prefers this epitope.

   I purchased the antibody from Kodak, I haven't tried any others... The
catalog number IB13025, item name M2 antibody.

Mary Beth Mudgett, Ph.D.
Department of Plant and Microbial Biology
University of California Berkeley

I tried to detect a flag tagged protein (transiently expressed) in A.
thaliana protoplast and it was a disaster. The antibody atleast from
Kodak was pretty lousy. We checked its affinity for flag peptide and the
affinity was extremely low. However, I believe if you have stably
transformed plants expressing epitope tagged protein it may give you a
decent result. Now anti-flag antibodies are also avialable from sigma,
santcruz etc, and these may be of good quality. Hope this helps,

RISHIKESH P. BHALERAO
Swedish University of Agricultural Sciences

I tried FLAG epitope in transgenic tobacco.  I used M2 monoclonal Ab to
detect a protein in which I inserted a Flag internally.  It seems to me
anti-flag M2 will pick up at least one non specific band around 100 KD in
Western blot.  I tried in both transgenic BY-2 cells and transgenic tobacco
(Xanthi) plants and the results were very consistant. I think if
you perform immunoprecipitation before Western blot, you might have a
chance to get clean background.

Jia Li
University of Missouri-Columbia






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