FLAG

[James A. Shaw]

The FLAG system is marketed by IBI.  We have been trying to use this system
for some time with little to no success.  Our approach which I hope to show you
is inherently flawed is to put the flag epitope as near as possible to the
N-terminus of OFG (our favorite gene) express in yeast and do westerns and
immunofluorescence and immunoprecip.  In our case the FLAG was by neccessity
2 or 3 residues from the N-terminus.  When used strictly according to the
mfgrs instructions it should be right at the N-terminus, which is easily
achieved if expressing in E. coli (through the combination of the ompF signal
and the enterokinase cleavage site on the provided vector--which of course
is not suitable for use in yeast).  Two antibodies are available: M1 which
supposedly requires that FLAG be at the N-terminus and M2 which is not supposed
to care where the FLAG is.  FLAG-OFG expressed in yeast has not been detectable
with either M1 or M2 by any means available (western (gel or dotblot),
fluorescence, or precipitation).  I would not recommend use of this system
in a yeast expression context, it might work well if you just want to get
a lot expressed in coli and go on.  But beware of advertising promises.

Andy Shaw
NIH/NIDDK/LBM
(301)402-3038
andyshaw at helix.nih.gov 

These are my opinions alone and should not be construed as any officialstatement of the HHS or the Executive branch as a whole.
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