Well, as most of the fluorescent proteins are either dimers or worse upon desired over expression, "for easy going" (sometimes even with the letter "m" in front of). The latter fact would yield in positive but often "wishful" FRET signal, especially when carefully limited number of controls have been designed to be " biophysically smart". Therefore, all the discussions around the Ro or J seem to be redundunt... You might recollect that the famous textbook experiments have been performed with free, soluble donor-acceptor pairs at concentrations far above physiologically acceptable limits.
...my two cents...
Vitaly
________________________________
From: Mark Cannell <[log in to unmask]>
To: [log in to unmask]
Sent: Sun, November 22, 2009 1:51:19 PM
Subject: Re: FRET Forster distance - Iain Johnson's table and others
Hi All,
I must admit to being kind of bemused by this obsession with theoretical FRET distances in cell experiments. In view of all the uncertainties in dipole orientattion (etc.) I think that all you can really say is that if FRET occurs the dipoles are very close together (a few nm). If it changes, they are moving (translation and/or orientation) or the local environment is changing (lipid<-->cytosol, another protein entering/leaving) -and you really don't know which, of any of these, is the cause. I think it very revealing when people talk about absolute FRET distances and their changes when they really don't know what is going on. Why do papers generally not talk about the possible change in conformation/orientation of a protein or even that that insertion/removal of a third protein may be responsible for a FRET change?
Cheers Mark
George McNamara wrote:
> Hi Lily,
>
> You can calculate Ro, as well as J* (spectral overlap integral - in many ways more useful than Ro) using the spectra in the PubSpectra xlsx file and the Szollosi&Horvath FRET Excel file, both in http://www.sylvester.org/AICF/pubspectra.zip
>
> I don't have the Excel matrices skill set to create a comprehensive FRET donor vs acceptor table, but perhaps you or someone on the listserv would like to do this - whoever does it could write the work up for publication in Biotechniques, Nature Methods, etc.
>
> George
> p.s. there is also a nascent FRET calculator option at http://www.mcb.arizona.edu/ipc/fret/index.html (3rd tab above the graph box) - Carl Boswell at UA might benefit from a financial donation to make the "Calculate FRET" button fully functional (or my choice of Alexa Fluor 488 and 568 might not have QY or another variable).
>
> p.p.s. Ro vs J* ... Ro depends on quantum yield and somewhat on refractive index which many papers do not mention. My understanding is that the RI(s) in an ~1 um diameter sphere surrounding the molecules is "sensed' (search Pubmed for Suhling refractive index for an entry point to this literature). RI 1.34 (water) or RI 1.5 (immersion oil? glass? solid protein?) which are clearly not correct for cytosol (which is ~1.38 for mammalian cells). It would be useful to define a normalized J* parameter that results in easy to compare values (much like GM units (10^50 multiplier) puts multiphoton absorption into a easy to read range, or QY * Extinction coefficient / 1000 makes fluorophore efficiencies easier to read.
>
> At 05:41 PM 11/19/2009, you wrote:
>> Hello list,
>>
>> A while ago (2005 to be precise) there was a list exchange regarding the Forster distance of FRET pairs. Iain Johnson from Molecular Probe (now Invitrogen) posted a link to his measured table of Forster distance for Alexa dyes. Of course the link is no longer valid. I am wondering if Iain could generously provide that information again? Also, does anyone know of other existing tables for the other FRET pairs?
>>
>> Thanks,
>>
>> Lily
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> George McNamara, Ph.D.
> Image Core Manager
> Analytical Imaging Core Facility
> University of Miami, Miller School of Medicine
> Miami, FL 33136
> [log in to unmask]
> [log in to unmask]
> 305-243-8436 office
> http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
> http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
> http://home.earthlink.net/~geomcnamara
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