Hi

You can try to get the new generation of gfp like proteins, for instance the mNeonGreen: 

https://greenfluorescentblog.wordpress.com/2013/04/30/new-and-improved-the-next-generation-of-gfp/

Anyway the brighnest of the GFP also depends on how much the folding of the fused protein modifies its fluorescent yield. I would try, if possible to fused the tag N and C terminal to the protein and compare.

Also have in mind that version like the turbo GFP (the pmaxGFP from lonza) while brighter than EGFP, it needs to dimerize to give signal so it is not the best option for fusion proteins.

You can also check this paper: A guide to choosing fluorescent proteins. 

Nathan C Shaner et al. 2005 in nature methods.

Cheers

By personal observation, I have the feeling that the pMax-GFP from Lonza looks brighter than EGFP, perhaps you can google it and look for the mutation that increases signal

hope this will help

JP

We're also detecting a protein with low expression level and it works really good with venus :-)

Hi

You can try to get the new generation of gfp like proteins, for instance the mNeonGreen: 

https://greenfluorescentblog.wordpress.com/2013/04/30/new-and-improved-the-next-generation-of-gfp/

Anyway the brighnest of the GFP also depends on how much the folding of the fused protein modifies its fluorescent yield. I would try, if possible to fused the tag N and C terminal to the protein and compare.

Also have in mind that version like the turbo GFP (the pmaxGFP from lonza) while brighter than EGFP, it needs to dimerize to give signal so it is not the best option for fusion proteins.

You can also check this paper: A guide to choosing fluorescent proteins. 

Nathan C Shaner et al. 2005 in nature methods.

Cheers

Hi Andrew,

I suggest you clone your protein of interest in a vector and express it exogenously in your cell line to verify that the tag is not affecting the expression/function/localization of your protein. It would also help you to set the optimal microscopy setting for the visualization of the specific fluorescent protein (signal/intrinsic fluorescence). Take a look at the fluorescent protein table in the Nixon Microscopy U website, there is a ton of info about different GFP variants including extinction coefficient and relative brightness compare to GFP:

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

Adgene has an impressive catalog of empty vectors for several fluorescent protein fusion of this proteins worth to take a look at.

https://www.addgene.org/fluorescent-proteins/plasmid-backbones/

Good luck with your project!!

Thank you all for your help. I will directly compare several fusions to see which one is brightest.

(Angela - excellent advice, I have already confirmed that an N-terminal fusion of GFP to my protein of interest can complement a knockout cell line so it behaves like wild-type again)

Hi,

if red fluorescence is OK for your purposes, you may consider tdTomato. It is the brightest fluorescent protein available. Also, it has longer excitation wavelength, which is less phototoxic for live cells studies. An interactive chart for selection of FP with optimal excitation, brightness and stability can be found here: http://nic.ucsf.edu/FPvisualization/

Hi,

brightness is only one property:

- pH dependency

- aggregation

- similiarity if you want to do simultaneous double transfections (Construct conversions caused by simultaneous co-transfection: "GFP-walking". Bestvater F, Knoch TA, Langowski J, Spiess E. Biotechniques. 2002 Apr;32(4))

- etc. etc.

all somehow play a role, and the difference in brightness, i.e. photon emission might not be so different in respect to the other influences.

so perhaps making a test, might not lead you to what you want and perhaps rethinking the experiment might be easier and might lead you further...

I agree with TA Knoch above, you need to consider also other properties of your FP in order to choose the best one. Photostability is quite a big deal, especially if you want to integrate your signal over time.

You might want to check out the review by Day and Davidson on Chemical Society Reviews 2009, and the one by Chudakov et al, on Physiol Rev 90:110-1163 (2010).

Both go quite into detail about the respective merits of different FPs all over the visible range.

Also, if your construct is poorly expressed, you might want to transfecr your POI-GFP cassette into a higher expression vector, unless you absolutely need low leves, in which case, ensemble imaging of your construct is going to be tough regardless of the fusion construct.

However, if you think your construct is poorly expressed because it doesn't fold really well, you might want to give sfGFP a go. It is from Aquorea and it is supposed to work quite well with tricky fusion partners. Shaner et al, Nat Meth 2013 report that it is brighter than garden-variety EGFP and as photostable. mEmerald is also supposed to be brighter than EGFP, but has a two-component photobleaching that might make your experiment trickier depending on the technique.

According to Shaner et al, J Cell Sci 2007, however, sfGFP can give you higher background because even misfolded tagged POIs still produce fluorescence, so be wary.

If you don't strictly need a GFP tag (which instead seems the case), you could try self-labelling tags like ACP/MCP tag or SNAP tag (the former only works with surface proteins, though).

They aren't much bigger than a GFP tag and can be labelled with normal fluorescent dyes, which are on average brighter and more photostable than FPs.

Bosch et al, Biophys J 107:803-814 (2014) is a handy guide to choosing which dye to use for intracellular SNAP tag fusions, while for ACP/MCP tag you can use Zanetti-Domingues et al, PLoS One 2013 (yes, I know, this one is mine) and Hughes et al, PLoS One 2014 as a reference.

I have tried ACP tag constructs, and the dyes follow pretty much the same behaviour described in those two papers (i.e. less hydrophobic dyes = less background).

https://en.wikipedia.org/wiki/SNAP-tag

https://www.neb.com/applications/cellular-analysis/acp-tag-and-mcp-tag-systems

http://www.ncbi.nlm.nih.gov/pubmed/16369541

http://www.nature.com/nmeth/journal/v10/n5/full/nmeth.2413.html

http://jcs.biologists.org/content/120/24/4247.long

Dear Andrew

We have compared a lot of fluorescent proteins both in vitro and in vivo. The best brightness was seen with the ZsGreen1. On the website of Clontech we have found that the brightness of ZsGreen1 is effectively 500% higher as those for GFP.

I hope this message will help you

Best

Pascale

Hi Andrew -

Another trick for enhancing detection of FPs with green emission spectra is to switch media during imaging. There can be a lot of background autofluorescence in cell culture, which seems to be due to riboflavin in the media. We have found that using a riboflavin dropout version of our base medium results in much better signal-to-noise ratio. There is now a commercially available base medium from Life Technologies (FluoroBrite DMEM) which performs comparably to standard high glucose DMEM in our hands, in terms of cell viability and proliferation. Leibovitz (L15) base medium has been used as a substitute by others, but we find that the composition is very different (galactose instead of glucose, for instance) and our cells do not grow well in this medium.

Therefore, you should validate that your cells behave similarly in a substitute medium before moving forward with live imaging. But the payoff in terms of detection of low signal is definitely worth it.

Cheers,

Brigitte

Hi Brigitte,

to get pH sensitive quality controls out of the medium is an old thing - so for any imaging take "colourless" medium.

actually that should be the standard...

Cheers,

Tobias

Phenol red-free media are very easy to get, and have been standard for live cell imaging applications, yes. Riboflavin-free medium is not as common, and until recently was a custom order from Life Tech, for example. Some cell types, such as human pluripotent stem cells, are radically altered when switching to a (more widely used) Leibovitz base, even when all other components and conditioning are identical.

well what anybody should actually do ist to make an excitation/emission analysis of the medium you use, phenol red, or riboflavin, or whatever is in there, then one really knows...

but you are right, Brigitte, Riboflavin as background is not on so many peoples agenda...

The answer to my own question: "mClover3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP derivatives" (link to paper attached).

http://www.nature.com/articles/srep20889

Sometimes the brightness may be tricky and depend on protein expression, degradation and other factors in a host-specific manner:

http://biorxiv.org/content/early/2016/02/19/040279

I will recommend to make the best use of the FPbase. It's a comprehesive website about FP.

https://www.fpbase.org/