Do you have any limitations for detecting the protein? Is this for an expression construct or are you labeling isolated protein? This is a good question for many looking to visualize a protein of interest but will depend largely on the laser/filter combinations available in your lab/department/core/university.

Agreed. eGFP is very bright, and most confocal or fluorescence microscopes will have the appropriate excitation and filters for green. If you want to eventually track more than one protein, or do FRET, there are other FPs that would nicely complement the GFP.

Hi,

From my experience I would advise super fast folding GFP, which in my hands gives by far the strongest signal. Although, a recent paper suggested that it could induce the formation of dots in cells, so you should have that into account. sGFP is good because it allows both C- and N- terminal fusions without much trouble which it is not always true for more common F. proteins. And if you don't have problems with using other systems we can try the SNAP-tag system that works very well in several systems.

Thanks for all your answers. the problem with EGFP and or turboGFP is that they are known to dimerize in particular conditions... I want to avoid that. Since we have the equipment for mcherry, i will test it first. I don't need to perform FRET either. It is just to label a shuttle protein.

Thanks again,

Thierry

Good luck!

I had the same question as yours. Maybe you want to study the interactions between proteins or their co-location, so you don't want to form dimers. I used the Ds-Red monomer as the maker. However, the protein has a big problem. It's fluorescent is too weak. Sometimes I can't figure out it from background. So I think the GFP is still the best chose.

If you find that your signal is not very strong or you have significant cell death there are several promoter combinations available commercially for mCherry. CMV promoters can often make toxic levels of your target protein so stronger is not always better. Best of luck! Keep us up to date on your progress with pictures.

Hi Thierry!

mCherry is monomeric and indeed seems to be quite stable (as for photobleaching), yet it has only about 50% of brightness compared to EGFP. This means that you actually might need to use more laser power/longer exposure and that at the end might contribute to photobleaching. 

As you noticed, EGFP, superfolderGFP etc. have a weak tendency to form dimers. However, a monomeric EGFP (mEGFP) has been obtained by a point mutation so it might be worth trying it (I am happy to give you the sequence). Another protein that is very stable and again twice as bright as mCherry, is mKO and therefore it is advised to use it when photobleaching is a problem. It is also believed to be a true monomer. 

Also, do you think you might use tandem repeats of a fluorescent tag for a better signal/brightness or will this perturb the function of your protein? 

You might find these resources useful:

http://www.nature.com/nmeth/journal/v2/n12/abs/nmeth819.html

http://www.microscopyu.com/articles/livecellimaging/fpintro.html

Another solution, as Pedro suggested, is SNAP-tag. The system has been extensively used in mammalian cells, yet here is a paper in which they are using it in live (I believe) bacterial cells:

http://mic.sgmjournals.org/content/155/6/1786.full

Good luck,

Michał

Is mCherry toxic to the cells?.eGFP is toxic and generally cells round off with gfp expression