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[–] BSL5 0 points 2 points (+2|-0) ago 

Yes, your concern is very real, and is highly dependent on the particular experiment being performed. The type of fluorescent tag used, how it's attached, where it's attached, and its size relative to the tagged protein can all interfere with function.

For example, in the case of a translational fusion (the tag is encoded in the DNA, so the ribosome produces a single protein-tag entity), the tag can impair the folding of the protein (and the tag itself), which can largely inhibit function (or fluorescence), and even lead to additional downstream effects if the unfolded peptides begin to aggregate.

Generally, control experiments will be performed to ensure that tagging effects are negligible, or at least to quantify the effect that the tag is imparting on the system. However, like anything in biology (and this is the part that keeps me up at night), there's always the possibility of some additional effect that it's having on the system that we don't know about. It could be negligible, but it could be completely screwing with your study. The more controls, the better.

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[–] jhawk1729 0 points 1 points (+1|-0) ago 

It can be a very real problem. Some standard controls include:

  • compare the localization of the GFP tagged protein with an antibody against the protein or an antibody against a very small tag added to the protein
  • compare cells expressing the GFP vs normal cells: do they grow at the same rate, do they have the same general morphology, do they perform a specific function (endocytosis, exocytosis, cell division etc) at the same rate as the normal cells
  • in many model systems (yeast, fruit flies, worms etc) you can use complementation to check for functionality: express the GFP fusion protein in an animal or cells that have the normal protein knocked out or knocked down. If the GFP fusion can rescue the phenotype caused by the knockout/knockdown, it's functional. In mammalian cell culture this is often done by using RNAi against the native protein and then changing that part of the protein (or untranslated parts of the mRNA) in the construct so that your fusion protein isn't knocked down

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[–] jhawk1729 0 points 0 points (+0|-0) ago 

It can be a very real problem. Some standard controls include:

  • compare the localization of the GFP tagged protein with an antibody against the protein or an antibody against a very small tag added to the protein
  • compare cells expressing the GFP vs normal cells: do they grow at the same rate, do they have the same general morphology, do they perform a specific function (endocytosis, exocytosis, cell division etc) at the same rate as the normal cells
  • in many model systems (yeast, fruit flies, worms etc) you can use complementation to check for functionality: express the GFP fusion protein in an animal or cells that have the normal protein knocked out or knocked down. If the GFP fusion can rescue the phenotype caused by the knockout/knockdown, it's functional. In mammalian cell culture this is often done by using RNAi against the native protein and then changing that part of the protein (or untranslated parts of the mRNA) in the construct so that your fusion protein isn't knocked down

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[–] jhawk1729 0 points 0 points (+0|-0) ago 

It can be a very real problem. Some standard controls include:

  • compare the localization of the GFP tagged protein with an antibody against the protein or an antibody against a very small tag added to the protein
  • compare cells expressing the GFP vs normal cells: do they grow at the same rate, do they have the same general morphology, do they perform a specific function (endocytosis, exocytosis, cell division etc) at the same rate as the normal cells
  • in many model systems (yeast, fruit flies, worms etc) you can use complementation to check for functionality: express the GFP fusion protein in an animal or cells that have the normal protein knocked out or knocked down. If the GFP fusion can rescue the phenotype caused by the knockout/knockdown, it's functional. In mammalian cell culture this is often done by using RNAi against the native protein and then changing that part of the protein (or untranslated parts of the mRNA) in the construct so that your fusion protein isn't knocked down