I am attempting to localize a FLAG epitope tagged protein product of
transiently transfected cDNA, into HEK cells, by immunofluorescens. While
I can see the one protein, expressed from the same vector, I don't see the
other. I know the cDNA is functional from in vitro expression studies, and
I know it has been taken up by the cells. I suspect the protein is simply
not expressed at high enough levels to be seen. IP from oocytes suggest
that is expressed relatively poorly there. I think I have exhausted tricks
to amplify the signal. So, what I need is a higher primary signal. I am
thinking of putting in multiple FLAG tags at the location, the n-terminus,
where I currently have one FLAG tag.
Anybody have experience with putting in several consequtive FLAG tags?
Does it actually improve the signal? If so, how much? How many epitopes to
add? Use spacers? If so, what should the spacers look like?
Christer Ericsson, DDS, PhD
State University of New York at Buffalo
Department of Biochemical Pharmacology
327 Hochstetter Hall-North Campus
Buffalo, NY 14260-1200
email: ericsson at acsu.buffalo.edu
phone: (716) 645-3926
fax: (716) 645-3850
Videoconference: CU-SeeMe IP# 126.96.36.199