Looking for a fluorescent protein.

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IronFish
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Looking for a fluorescent protein.

Postby IronFish » Sun Mar 25, 2012 1:22 pm UTC

Hi, I'm looking for a monomeric small fluorescent protein.
The smaller the better.

Any suggestions?

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Re: Looking for a fluorescent protein.

Postby ahammel » Sun Mar 25, 2012 3:11 pm UTC

What for? Smaller than GFP (238 residues, 26.9 kDa)?
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Re: Looking for a fluorescent protein.

Postby Izawwlgood » Sun Mar 25, 2012 3:28 pm UTC

A bit of poking around doesn't even reveal any intrinsically autofluorescent proteins. I think you're going to be limited to GFP varieties (which at 26kd is pretty tiny) or quantum dots.

What's your goal?
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Re: Looking for a fluorescent protein.

Postby Gigano » Sun Mar 25, 2012 3:58 pm UTC

Some of the monomeric fluorescent proteins I know are:

Red:
mCherry (236 aa)
mStrawberry (234 aa)

Orange:
mOrange (236 aa)
mKO (218 aa)

Yellow/green:
mCitrine (259 aa)

Green:
GFP (238 aa)

Cyan
mCFPm (~239 aa)
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Re: Looking for a fluorescent protein.

Postby IronFish » Mon Mar 26, 2012 7:50 am UTC

Thanks for the elaborate answers!

I'm looking for a small protein to minimize it effects on its fusion protein (in particular wish to avoid disrupting its oligomerization and binding to other protein partners).

We already have the mFruit series and mGFP and its derivates.

I wonder if an FP<100 aa was developed (I'm willing to sacrifice brightness and photostability)...

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Re: Looking for a fluorescent protein.

Postby Izawwlgood » Mon Mar 26, 2012 1:05 pm UTC

Like it was said, fluorescent protein markers don't get really get smaller than GFP. If you're tagging something that oligomerizes and you're worried you'll change it's behavior, you just fiddle around with where you insert the protein. Considering actin is tagged quite successfully, I'm sure you can figure something out.
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Re: Looking for a fluorescent protein.

Postby Ulc » Tue Mar 27, 2012 11:10 am UTC

Izawwlgood wrote:A bit of poking around doesn't even reveal any intrinsically autofluorescent proteins. I think you're going to be limited to GFP varieties (which at 26kd is pretty tiny) or quantum dots.

What's your goal?


I'm obviously too used to my particularly subfield when my immediate thought is "26kd? That's like, gigantic".

For reference, I work with a 4kd peptide currently and my last project was a 11kd presumed monomer (it probably wasn't monomeric).

IronFish - I doubt that you're going to find any <100 aa peptides that are fluorescent, it's not enough residues to form the core that folds around the photodyes. But have you considered stealing a page from sm-FRET*? And uttilising the Alexa dyes that they use - they are attached chemically post-translational, so it should be fairly easy not to put them in a protein-protein interaction site.


*I have my own personal doubts about sm-FRET as a technique I might add, it's a cool and awesome technique, and the results are impressive, but I'm a bit suspicious about any method that assumes that attaching large non-polar dyes to a relatively small polar protein doesn't change the dynamics grossly - particularly when said dynamics are exactly what you're aiming to study.
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Re: Looking for a fluorescent protein.

Postby Izawwlgood » Tue Mar 27, 2012 12:25 pm UTC

You don't work on whole cell systems I take it.

And yeah, FRET is a little hocus pocus for my tastes. There are better techniques for showing interaction.
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Re: Looking for a fluorescent protein.

Postby Ulc » Tue Mar 27, 2012 1:01 pm UTC

Izawwlgood wrote:And yeah, FRET is a little hocus pocus for my tastes. There are better techniques for showing interaction.


I like sm-FRET, it's an awesome method for detecting single molecule dynamics and conformations - working at ensembles of protein has been the MO for the last 60 years, with no methods providing really in-depth knowledge of micro-populated states (something we only really during the last decade has come to understand is hugely important). I'm just not sure that I trust the method all that much.
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Re: Looking for a fluorescent protein.

Postby Izawwlgood » Tue Mar 27, 2012 4:19 pm UTC

It's a neat technique in theory, but my experience with is that there are so many confounding and complicating factors that it is nearly useless. You want to show colocalization? Just use two separate fluorophores. You want to show interaction? Do it in vitro. Having two honking fluorophores that must interact with one another to complete the experiment seems a pretty large assumptive hurdle to get over.
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Re: Looking for a fluorescent protein.

Postby Angua » Tue Mar 27, 2012 5:16 pm UTC

However, I have seen a couple of papers where they use that technique (interaction leads to ability to fluoresce). So if you want something really small it might be useful, but I don't really know much about where exactly you have to insert the things into the two respective proteins so they interact.
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Re: Looking for a fluorescent protein.

Postby Izawwlgood » Tue Mar 27, 2012 5:41 pm UTC

I think the trouble is that there are better assays to show interaction (not just co localization) and perfectly sufficient assays to show co localization. FRET is pretty finicky, and doesn't really answer anything you can't answer more or just as assuredly with more reliable tests.
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Re: Looking for a fluorescent protein.

Postby Ulc » Tue Mar 27, 2012 11:51 pm UTC

Izawwlgood wrote:I think the trouble is that there are better assays to show interaction (not just co localization) and perfectly sufficient assays to show co localization. FRET is pretty finicky, and doesn't really answer anything you can't answer more or just as assuredly with more reliable tests.


But there isn't better assays to show single molecule interactions, dynamics and kinetics. in fact, sm-FRET is pretty much the first non-ensemble method available, and studying ensembles are what we've being doing for the last 80 years, sm-FRET is awesome for that, even if the results aren't perfect.

I mean, the method is literally studying one single molecule at a time! That is unbelievable awesome!
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