(by ucgatan from ucl.ac.uk)
Fri Feb 29 06:28:24 EST 2008
On Fri, 29 Feb 2008, Aawara Chowdhury wrote:
> 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.
Although this does vary from protein to protein - i have a myosin
II antibody that gives quite a bit of cloudy background on top of the
signal, and a GFP fusion that gives a breathtakingly clean localisation.
Of course, it's possible that the cloudy background is something
biologically significant that the GFP is for some reason missing, but i
sincerely hope not!
There have been papers on the effect of GFP on localisation; i've read
some of them, but can't for the life of me find them now - try
pubmedicating for 'GFP' and 'localization', and weep! All it means,
really, is that you need to validate the localisation of a GFP fusion
against antibody staining before you draw any conclusions from it. And, of
course, the antibody staining needs to be validated somehow - as well as
the usual blot-based tests, staining a cell in which your protein is
knocked out or down is the gold standard.
Note that in the case of some fluorescent proteins, there's also a risk of
oligomerisation artifacts: wildtype FPs (all or just some) are tetrameric,
so if you attach them to a protein, you're adding a tetramerisation
domain. Modern versions have been engineered to be monomeric - that's what
put the m in mRFP - so this is not a worry, but if you have some crufty
old construct that's tagged with DsRed, watch out.
There is a relatively new alternative to GFP for fluorescent tagging of
proteins in live cells in the shape of the FlAsH/ReAsH system:
Basically, you put a magic tag on your protein, and then add a
membrane-permeant fluorophore that comes in and binds specifically to it.
The tag is only six residues, so it doesn't interfere with behaviour as
much as GFP, and the fluorophore is only active once bound to the tag, so
it's specific. Plus, you can do funky pulse-chase experiments to look at
dynamic processes. At least, that's what the marketing bumph says. I've
heard that the fluorophore solution makes cells unhappy, and there's quite
a lot of background. It's a fairly new method, though, so it's probably
improved a lot recently. It's rather pricey, though.
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson from ucl.ac.uk
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