that's funny Simon, I had just the opposite case in yeast, where td-tomato caused some untypical behaviour at tagged proteins...

One point which wasn't mentioned yet, is the maturation time. mCherry is quite fast, comparable to GFP variants, but most other RFPs have at least one hour maturation half-time. So, if you are interested to see changes in localization or responses to different cues, which you expect to be fast, then use mCherry, it works even nice as threefold-tag for us but is not as bright as other RFPs. If you want I have some nice papers which list the maturation times and photostability of different fluorophores, but I'd have to search them first.

Dear Jonas

It'll be important to remember to check which version of RFP you're using. Some versions of RFP or dsRED form tetramers so I'd advise to check how useful that would be for your experiments.

There are monomeric alternatives such as mCherry and a monomeric dsRED by clontech. All the vector data and fluorescent spectra are available on their website.

I have used dsRED in COS cells with good localisation results. However, I found it sensitive to bleaching so it may be helpful to keep the laser power low.

Cheers

Andreas

RFP is quite sensitive to photobleaching. If you are not too picky with the type of red fluorescence you want, mCherry is also easily available from other labs for free and is more stable and brighter. There are however some other excellent red fluorophores from orange to far red, some of them available from the Tsien lab with a MTA, or you can buy them from various companies such as Clontech or Evrogen. Check on Addgene first as it is only 65 bucks per plasmid.

I would concur that mCherry is in a general a good choice. However, you should be mindful of which laser line you have on your imaging system (e.g. 543 nm, 561 nm, 568 nm), as well as the emission filters; the red FPs differ in their excitation and emission maxima (e.g. http://www.clontech.com/GB/Products/Fluorescent_Proteins_and_Reporters/Fluorescent_Proteins/Fusions/Red).

I should also point out that in some cases we have seen aggregation of mCherry-tagged proteins in fly neurons under conditions when tdTomato-tagged equivalents do not aggregrate.

that's funny Simon, I had just the opposite case in yeast, where td-tomato caused some untypical behaviour at tagged proteins...

One point which wasn't mentioned yet, is the maturation time. mCherry is quite fast, comparable to GFP variants, but most other RFPs have at least one hour maturation half-time. So, if you are interested to see changes in localization or responses to different cues, which you expect to be fast, then use mCherry, it works even nice as threefold-tag for us but is not as bright as other RFPs. If you want I have some nice papers which list the maturation times and photostability of different fluorophores, but I'd have to search them first.

Dear Jonas, just a comment on ur imaging setup n subcellular colocalization analysis. Be mindful that even w an ideal FP u will still need super high resolution images if not nanoscope images for good subcellular colocalization...

Hi,

We used RFP for labeling Treg cells and it was a pain to see them under the confocal. If you need to use flow cytometry make sure they have the right filters and on top of that is not very bright. I would recommend to look first at the tech specs of the instrument you want to use and choose the fluorescent protein according, so you will get best results. Good luck!

In principal, in my own experiments for colocalization I always use an RFP to tag something for what I know localization and which is constantly expressed and localized (organelles, centrosomes etc.) and than I'm using GFP variants for the actual subject of the study as normal GFP is around five-times brighter than mCherry and maturation is also of the same range faster as for brighter RFPs like td-tomato. Furthermore, GFP filters and spectra don't differ this much as those for the red spectrum. So maybe you should change your concept to get your results with less effort and frustration.

I concur that mCherry or Tag -RFP -T are good options. Just to be contrary have you thought of labeling you viral proteins without making XFP fusions? I would suggest looking into another Tsien lab creation that has been used successfully to label viral proteins namely the tetra cysteine tag and the FlAsH, ReAsH or lumio biarsenicals. I am attaching a link that might help get you started if you are not already thinking in these terms

http://www.sciencedirect.com/science/article/pii/S1046202311001666#

Good luck

Yep, check your laser lines and go for mCherry. Rfp photobleaches easily, and I never found it to be as bright as mCherry

Fluorescent proteins often exist in several forms in vivo, subject to their concentration and intracellular conditions such as pH, presence of lipids, etc. Partial unfolding and especially oligomerization can significantly alter photophysics of RFP just as most other FPs as they find their way within cell through a series of organelles. So to answer your question, AT THE VERY LEAST, I would investigate literature for performance of RFP in the organelle where your viral protein is expected to be delivered.

Also imaging method is of essence here, for example excessive blinking can be an issue in FCS while non-exponential fluorescence lifetime decay shall be an obstacle for FLIM and FRET imaging.

RFP naturally forms tetramer, so that it is a bad choice to track proteins. mCherry is a good option.

The wisdom in the lab I did my doctoral research in is that it's much safer to GFP-tag a protein, as GFP is less susceptible to photobleaching than the RFPs we worked with. I can personally say that as a cell fill, RFP was indeed more likely to bleach during time-lape imaging than GFP fill.

Based on my experience, mCherry is definitely a better option (over other RFPs) in case you want to use dual-labeling for co-localization studies. However, as Christian Renicke pointed out: it's not as bright as other RFPs. Choose accordingly!

mCherry is brighter than RFP, but the literature suggests that mRuby is one of the brightest versions of RFP.

If you are looking at fixed cells and want to increase signal via antibody staining, the best anti-RFP antibody I have found is the rabbit polyclonal from Rockland.

Brightness and photostability issues aside, mRFP has some interesting advantages over GFP. For one, it is better suited for proteins situated in acidic compartments than GFP, which displays weak fluorescence under such conditions. We have also found that GFP (or cYFP, which is a variant of GFP) can interfere with functional domains of the tagged protein, whereas identical fusion contructs made with mRFP are fully functional. Thus, we now always consider using RFP as an alternative to GFP.

I routinely image with mCherry on a spinning disc. The only issues I have found thus far was tagging a protease and a Rab6 -they function completely differently to their eGFP counterparts. I've had friends use TagRFP who really like it. I tried mApple- was no different to mCherry in my hands. Avoid DsRed as the immature form is green and can interfere with co imaging of other green flurophores (and it forms tetramers).

If you go with the FLASH/ReASH any point, I have it on recommendation that Toronto chemicals sell the reagent for a fraction of the price to invitrogen and it works.

Thank you all for the helpful comments and suggestions. I wasn't expecting such a deep discussion. It opened my eyes to some details about tagging proteins with FPs that I wasn't aware.

Anyway, I think I'll stick to the idea of using a red FP and, based on your comments, mCherry seems to be best choice (until the moment it proves not to be).