How to choose between flag, tag or fluorescent prot fusion for subcellular local - (Jul/13/2006 )
Hello, I would like to know what is the best flag or tag or fusion in order to study subcellular localisation of our protein of interest and its movement when we coexpress it with some other candidates.
We are curently using GFP and DsRed mono but since those prot are big, would an HA tag or Flag, or 6xHis, or Myc be better? I have seen some articles talking about decrease of protein expression or stability when tagged with HA, and the GFP seams to affect folding and stability and localisation.
Any advise??
Sincerly
J
In one of our studies we did find some instability when we tagged HA to the protein. But most researchers do agree this is alsways the case with HA tag. So U could try with HA and if it doesnt work, then go ahead with some other tags. We used AU1 tag, which was nice but sometimes had trouble with western blot. Once u get the western going, then its not a problem with AU1 tag.
Myc and Flag tag gave us lot of background especially for invivo studies.
Myc and Flag tag gave us lot of background especially for invivo studies.
Thanks for the info. sorry for the delay, 14th of july is off here!
I just started my post-doc in the lab and they are using GFP and found some mislocalization; since I am working with 2 new proteins I would like to try a different tag to look at their localisation via confocal microscopy, I already have AB which work for WB. Is the AU1 tag OK for confocal microscopy? what about background level? you say that myc and flag gave you too much BG for in vivo studies, are you using AU1 tag?
Did someone try the new system from promega with cloning off the protein of interest in frame with a non fluorescent tag and then you add a fluorophore green, red or blue , which avoid cloning into pEGFP and pDsRed etc...?
Have a good week end.
Juliette
AU1 never gave any background. In fact its good for immunocytochemistry. For western, u have to optimise it. Thats all. We used AU1 in all our protein constructs in my old lab.
This is the paper. u can check it.
Evaluation of epitope tags for protein detection after in vivo CNS gene transfer.
Eur J Neurosci. 2006 Apr;23(8):1961-9.