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James A. Shaw andyshaw at HELIX.NIH.GOV
Tue Jun 22 09:04:42 EST 1993

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
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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