Hi Daniel,

my experience concerning subcellular localization of GFP-tagged proteins was usually pretty bad. Since GFP is located on ether the N- or C-terminus and has a size of appr. 26kDa, it can influence subcellular localization a lot. Overexpression influences localization as well. One option could be to use less DNA for transfection, to avoid artifacts produced by strong overexpression. If you have a selection marker available you might also consider to make stable clones with low expression levels. But if you don't want to do live cell imaging, the use of antibodies directed against the endogenous protein might be an alternative.

Best Kai

Hi Daniel,

my experience concerning subcellular localization of GFP-tagged proteins was usually pretty bad. Since GFP is located on ether the N- or C-terminus and has a size of appr. 26kDa, it can influence subcellular localization a lot. Overexpression influences localization as well. One option could be to use less DNA for transfection, to avoid artifacts produced by strong overexpression. If you have a selection marker available you might also consider to make stable clones with low expression levels. But if you don't want to do live cell imaging, the use of antibodies directed against the endogenous protein might be an alternative.

Best Kai

Kai raises some good points. Both the size and relative stability of GFP can interfere with subcellular localization especially in membrane compartments. Moreover, overexpression of a protein, with or without GFP tags, can overwhelm normal processing or saturate protein binding partners leading to cytosolic accumulation or in some cases ectopic targeting to other subcellular organelles.

On the converse end, very low expression levels can lead to problems with cellular autofluorescence, which occassionally gets misinterpreted as improper localization of a GFP fusion protein.

I agree with Kai that you should try to demonstrate proper localization of the endogenous protein first, using indirect immunofluorescence. If this works, then you can proceed to see whether at lower expression levels you can obtain the expected distribution of the GFP-tagged protein or whether an epitope-tagged based approach might work better.

Kai and Daniel answered all your concerns. From my end, it is good to use..

1. Specific gene promoter to drive the GFP expression.

2. The stable cell lines expressing low copy number recombinant

3. If you want to take good immunofluorescence images, you need to acquire images in a linear range of green fluorescence and using two photon laser confocal microscopy or spinning disc confocal miscroscope to get good z-sections.

This will also prevent bleaching of GFP.

Hope this will help.

Hi, Daniel, I agree with previous comments, but want to add some minor points: you should also calculate how is your GFP tag will alter chemical properteis of native proteins and try to minimise such disturbtion during GFP-fusion contruction. How specific is your filter set? It is alos importnat to developmental stage of the cfells. Some type of the cells at certain developmental stage have a high autofluoresnce, but in a region slightly different from GFP maximumj. So, specifci filtre set will help to avoid high background, as well as optimal developmental stage.

If your expression is too low to compare with background, you can fix cells and perform immunolocalization against anti-GFP antibody. In many case you will have higher signal.

Good luck!

I do have in mind raising antibodies to my protein, a very expensive venture. I was just thinking l could use GFP as a proof of concept. Is there any fluorescence reporter which works better than GFP and gives less problems?

Hi, Daniel, I think antibody is a best option. You will spend time and money to get it, but thereafter you do not need to made transformation each time, can easy study protein co-localization with 2-3 other priteins and even study protein-protein interaction by using PLA assay. Good luck!

Daniel- you could try a flag or Myc epitope tag in lieu of GFP. Another option is the HaloTag, which can be directly conjugated with fluorophores.

Hi Daniel,

I assume you're using the pRPa system for GFP tagging in T. brucei? As this integrates at a rDNA spacer sequence, and the integrated expression cassette is driven by a rDNA promoter, expression is likely to be significantly higher than the endogenous protein; this could be causing the apparent mis-localisation.

So, you might want to try incorporating your tag at the native locus (you could use one of my pNAT constructs, or one of a number of other very good plasmids developed in Mark Carrington and Tom Seebeck's labs, which do the same thing). These replace one of the native alleles with a tagged version, resulting in tagged protein expression at closer to native levels - you should note that you'll almost certainly need to use an anti-GFP antibody to detect your tagged protein due to the low expression.

Are you using an anti-GFP antibody at the moment? As you're seeing whole cell labelling, this could be autofluorescence, and using an anti-GFP antibody may reveal your tagged protein - it's not unusual to be unable to detect GFP-tagged proteins by direct fluorescence in T. brucei. You shouldn't need to use a deltavision system to get a basic localisation, so get the localisation working first, then use deltavision if it's indicated, e.g. if you want to get some cool 3D realisations - afterall you can't make your protein have a cool localisation!

Another thing you should consider is putting the tag at the other end of your protein - localisation signals are often found at the N-terminus, so tagging at the C-terminus can often solve the problem. Hope this helps - if you can provide any more specifics, I might be able to make some more suggestions - good luck with your localisation.

Thanks to everyone. I am presently not using anti-GFP antibody. I will consider using that.

Yes Sam, l am using the pRPa system. I believe l could lay hands on the pNAT somewhere in the lab. Since l intend to generate antibodies to the native protein, do you suppose that will be better in determining localisation in situ. How good is the pNAT with anti-GFP antibody in imaging localisation. Could try that whilst l wait for my antibodies to be made.

Hi, Daniel, antibody against native protein is always much better to compare with anti-GFP one. The reason has been mention in previous posts: behavior of recombinant protein is not always follow behavior of the native one in the term of folding, targeting, trafficking. These differences between native and recombinant proteins were largely dependent from the nature of your protein of interest: how strong GFP-fusion change protein properties and localization, in which part of the native protein FP was inserted, stage of cell development etc.. Good luck!

Hi Daniel,

I'd recommend using the anti-GFP antibody from Molecular Probes (part of Life Technologies), it's worked well for me - I think that only about a quarter of the proteins I've localised in T brucei have been detectable by direct GFP fluorescence. Raising antibodies against your protein will probably be expensive and time consuming, and though they can give a definitive answer, there's no guarantee of success. So, you'll need to balance the pros and cons of either approach to localising your protein - you can try a few basic things (as in my previous comment) then decide whether it's worth trying to raise an antibody. Regarding pNAT, it contains the same GFP sequence as pRPa, so the only difference is the likely level of expression (~native vs RNA pol I driven over-expression). Hope this helps

Coming from the plant cell/live imaging field I would only like to add a few minor points to the discussion.

1. In my opinion, using a GFP antibody when you see the GFP signal everywhere in the cell will most likely not change the result. But a smaller tag (like the above mentioned Myc-tag) might improve your results.

2. My second point is more a general one regarding the protein itself. A xFP often changes the localization: have you tried both, N- and C-terminal tagging?

(we observed this when we put xFP to SNARE proteins) Furthermore you have to consider that the GFP could mask signal peptides or other recognition/processing sites, especially in the N-terminal region.

3. Due to my second point, I usually use a small linker (6aa) between the xFP and the protein of interest. You may also consider that a directly fused GFP might impair proper folding of your protein... This could also be avoided by a linker.

Did you try confocal microscopy?

Hi Falco - it's very easy to confuse the slight green haze seen in T. brucei in the 'green' channel with real GFP expression, especially if the exposure time is long (or if you want to see something), hence my suggestion to use an anti-GFP antibody (obviously, the appropriateness of this approach depends on the brightness of the signal that Daniel is already seeing).

I certainly agree with your comments regarding N- or C-terminal tagging (as per the last para of my previous overly long comment), as well as the suggestion to use a smaller tag made by yourself and DC. The expression construct that Daniel is currently using includes a 2-aa linker between the GFP and the tagged protein, extending this might be worth while, but I'd try the other alternative approaches first, as these tend to work in most cases that I've encountered with this set up in T. brucei.

Hi, Sam, in "green channel" signal is largely dependent from filter set you have used. In the case of epi-fluoresecnt microscope with less specific filter set I often observed high green background. However, with more narrow filter set, or in the confocal microscope this background do not visible anymore and only specific signal appear. Anti-GFP antibody apparently increasing signal, especially if you use primary and secondary antibody. In the case of Dy-Light antibody the signal apparently very bright, much more stronger than GFP.

Hi Daniel, I too agree with the above….

@Sam

Only for my information, I have a good experience with several model organisms but I have none with T.Brucei. So, my question is how do you target only one allele? Is it only by chance and you still have to screen individual strains? Also, if T.brucei doesn’t have non-homologous recombination then it will be far easy for Daniel just to PCR the GFP tag in your pNAT plasmid or any other one using proper homology oligos and integrate the cassette in frame just before the stop of his Orf. I guess there are other tags which he can use too with pNAT, etc…

In case this point hasn't already been hit on in the thread, if you choose GFP-based imaging, Daniel, you should try to perform it in live cells. Fixation can quench GFP and make it more difficult to observe proper localization in cases of weak expression (that is, assuming your cytosolic GFP is really just auto-fluorescence).

Of course, live cell imaging might be a bit tricky for motile cells such as T. brucei, but perhaps Sam knows some tricks to transiently immobilize them. In some experimental systems, you can trap cells in viscous solutions or hydrogels to limit their mobility for the purposes of imaging.

Have you confirmed that your fusion is intact using Westerns? There are reports of GFP fusions beiing cleaved, which could lead to a result similar to what you are seeing.

Hi Daniel,

is your protein functional? you should check this because tagging proteins can influence their function and subsequently their cellular localization, do you have any way of checking if it is functional?. If you have only one copy gfp tagged you can have protein cleavage and the signal you are seeing is the GFP alone. What you can do is purify cell extracts and run a gel to see if the size is correct, I can get you a protocol for this. GFP can five false results, but in my experience is not worse than any other fluorescent protein, if you want to change I would advise the SNAPtag (check the New England Biolabs page for details on the SNAPtag),it has been shown to give less problems but again it has some problems. You have to use a dye to label your protein so it is less direct than GFP and it is apparently very good for E. coli and B. subtilis but I work in S. aureus and it is not very good.

Good luck if you need more information just tell me.

What George just said - meant to mention this in my earlier answer, but completely forgot! This is really the first step in any trouble shooting of the tagging - and indeed, is something that I normally do before I even look at the cells.

Taras - indeed, you're right, the degree of apparent background is influenced by the filter set. I'm really curious to know how bright the 'GFP' signal is that Daniel's seeing; this will ultimately determine whether or not it's worth using the antibody to detect the 'real' signal.

Regarding live cell imaging (DC's comment), this is certainly possible in T. brucei, and there are a number of protocols, but people routinely use fixed cells, as it's considerably easier (though as you say formaldehyde-quenching can be an issue, but there are alternative fixation methods, such as ice-cold methanol).

Abdelhalim - T. brucei carries out homologous recombination, so we're able to target specific regions of the genome with our constructs, though which of the alleles is targeted (T. brucei is diploid) is random. We've also used PCR to generate integration cassettes, but this approach has been limited to the generation of knockout or reporter cell lines (PCR amplify the selectable marker, including UTRs from a plasmid, including specific targeting sequences in the primers). Sorry off-topic, but thought I should try to answer the questions.

Hi Sam,

Thanks! Although it is a bit off topic but RG threads are meant for this too, if its works for knockout then you can engineer a plasmid which has gfp tag or other tags just before the clonat or other resistance markers and it should work. That will make it easy for what Daniel intend to do also.

Thanks everyone. l have not used the Deltavision yet. However, although the green filter seems to show in the background, you coud definitely see that the cells are fluorescing with the GFP. You could also see points of higher fluorescence than others. As to whether these are real or not, l hope to find out. l guess using live cells will answer some of these questions. Please see attached picture. Might not be the best but should give an idea

Just so l know my proteins are working, l have the same proteins over-expressing without GFP tagged and l do see growth and biochemical defects. So yes the proteins are functional-Pedro.

Hi Daniel - from the image you just posted, I don't think you need the antibody! Given the fluorescence is so strong, it might be worth going for tagging the native allele with a pNAT plasmid (or similar), as this will remove the potential effects of over-expression. Also, you should definitely do a western (if you haven't already done so) to confirm that what you're seeing reflects GFP-fusion protein localisation and not just a a fragment thereof. I would also repeat what I, and others, have said earlier in this thread, that you should be clear that you're not disrupting any localisation/signal sequences with your tag, ie you may need to tag the other end from the current set up. Finally (for now!), if there are points of higher fluorescence, these may be clarified by confocal microscopy and/or deconvolution. Good luck, it's looking promising!

Hi, Daniel, you have a very strong signal! So, just try to set you imaging, may be with confocal picture, and, if you use fixing cells, just mounting in DAPI-GOLD antifage medium. In this case you will have idea about protein location in the cells becuase you will have nucleus as a marker. Good luck!

For the Sam: methanol fixation is quite dangerous for FP fluoresence, Formaldehyde fixation is 100% saving for FP. Moreover, auto-fluorescence from formaldehyde can be reduced by several washing after fixation and, also, can be separated from GFP by appropriate excitation/emission set.

Thanks Sam. What do you mean by tag the other end? Are you suggesting trying a C-terminal instead. l have to check the pNAT we have to be sure.

Yes, exactly that, there are both N and C-terminal variants of the pRPa and pNAT plasmids (see Alsford et al, 2008, MBP for details). Are you doing the work in Glasgow? If so, the labs in the Wellcome Centre there have most of my constructs, as well as some variants that they've made themselves. Otherwise, drop me a line when you know what you need and I'll put it in the post.

I still think you should do an anti-GFP western first though, just to ensure that the tagged protein is the correct size before you start doing any more cloning - this will also show whether you've tagged the right end, then you can design your pNAT tagging strategy accordingly (if you decide to do this).

Taras - As you suggest, I also normally fix in PFA for fluorescence and immunofluorescence, which works fine with plenty of washing. I've also fixed GFP cells in methanol for FACS analysis and been happy with the results. So, I guess the protocol used is a decision made on a case by case basis.

Hi Daniel

As i told you earlier, just wash the cells with gently fixing cells with 2% formaldehyde for < 10 min at room temp and use low concentrations of DAPI (less than 10 mM) to counterstain the nuclei and wash it three times with 1x PBS and take Confocal images with green laser, this will resolve your problem. The Pic what you posted have high background becoz, they are not acquired properly and not a confocal image. Hope this will help

RKG

Taras-What excitation/emission set do you use for GFP when using formaldehyde. Thanks

Hi, Daniel, for GFP you can use 488 excitation and BP 505-530 filter for emission. Laser power, detector gain and amplifier offset you should set experimentally. Do not forget to include negative control, ea. similar cell without GFP to set background. Good luck!

Just a small point: my experience suggest that organic solvent like methanol have a severe negative effect on florescent proteins. So, try to avoid methanol fixation, despite in some case it may not crucial as Saw mention. You can find more information under this link: http://microscopy.duke.edu/sampleprep/if.html

Hi Daniel,

Your problem may not be with microscopy at large even though a small additional work like deconvolution can define highly concentrated fluorescent loci in the cell. Problem probably lies at cloning step. Interestingly, GFP has high half life varying from few hours to few days depending on cell type. But, a lot of GFP variants were made to play with half life. Good size of literature is available on this aspect. So, one can choose a GFP variant close to the half life of protein of your interest. Since the GFP tag affects the half life of the resulting fusion protein, it spills all over the cell if it is constitutively expressed. One way to overcome this problem is observing cells short periods after transfection. On the other hand, if you loose the tag by signal peptitase during protein processing, a simple western blot will definitely show you the problem. In this case, I would do western blot and use antibodies specific to GFP and the protein of your interest.

Good luck with your troubleshooting.

Overexpression can result in artifacts. You can circumvent that by tagging in the genome or using a genomic clone such as a Bacterial Artificial Chromosome.

Also, maybe the tag is cleaved away from your protein of interest. You need to confirm by Western blotting that cleavage is not happening.

I really think you have a very high background fluorescence. Try solving that with proper microscopy setting and then work on other methods.