(by aawara from pontiff-playground.org)
Thu Feb 28 21:19:29 EST 2008
In <mailman.849.1204229789.2451.methods from net.bio.net>,
Yvonne Couch <yvonne.couch from dpag.ox.ac.uk> wrote:
> We have a bit of a debate raging in our lab about the effects of GFP
> tagging. Is it better to express a protein with a GFP tag in a plasmid to
> investigate its function/location/aggregation or to just express the protein
> alone and then use antibodies to look at it? There is some contention as to
> whether GFP itself might aggregate and therefore confound results. I
> personally think all the cloning required to get GFP out of one vector and
> into another is just too much hassle!
Depends on what your goals are. The advantage of a GFP chimera is that
you can follow the protein's dynamics in live cells, permitting the
study of environmental perturbations that affect localization, aggregation,
or even turn-over. You've pointed out the disadvantages already.
In my experience, indirect immunofluorescence gives "cleaner" results
for localization than a GFP chimera in fixed cells, particularly when
confocal microscopy is used, or image stacks are deconvolved.
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