Naked Science Forum
Non Life Sciences => Chemistry => Topic started by: Variola on 26/05/2009 21:10:26
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Hello all [:)]
Does anyone here know anything about FRET? ( Fluoresence resonant energy transfer??
I am doing some coursework, and despite my best efforts I am struggling with the equation. We had half a hurried lecture on FRET and my lecture slides are not clear!
This is the question I have been given:
The fluorescence resonance energy transfer efficiency measured between a rhodamine dye molecule and a cyanine dye on either end of a 15 base strand of DNA is E = 75%
a. Given that the 50% transfer distance between the two dyes is R0 = 60 Å (Angstroms) what is the separation of the two dye labels on the DNA.
And this is the equation we were given to work it out.
E= R06
R6 + R06
While I understand what Ro is, I cannot grasp what Ro6 is! So I have no idea how to rearrange the equation to get the separation number Google has so far been quite unhelpful and now I am really quite stuck. Any help would be really appreciated!! [:)]
EDIT: Just realised I hadn't set this up as a question. Oops!
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Ah, a few years ago I spent what at the time felt like a lifetime in a darkened room doing FRET and the like on my little fibroblasts. And although the confocal was programmed to do much of the grunt work, I really should be able to answer that. Will go away and try and remember what we did!
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OK, so I mostly did FRAP and FLIP, but did do some FRET. Can't really remember a lot, but my incomplete garbling may help fill some gaps somewhere!
R0 is the distance between the donor and acceptor probes where teh energy transfer is 50%. The equation is generally written with the '0's being subscript and the '6' being superscript. I guess this is something to do with the fact that 10 Angstrom = 1 nm, so the 60 Angstrom distance between the probes is 6 nm (but don't quote me - I'm working on it!).
E = R06/(R06+R6)
This graph is quite good:
(https://www.thenakedscientists.com/forum/proxy.php?request=http%3A%2F%2Fi143.photobucket.com%2Falbums%2Fr133%2Flindsayjemerson%2FForsterdistance.gif&hash=2647ccd727886c47e5cb5d47bdd20f8c)
So, I think essentially, in this case:
R06= 60
E = 0.75
You can re-arrange the equation as follows: (I'm not doing sub and superscript anymore, its getting annoying!)
E(R06+R6) = R06 (multiply both sides by (R06+R6))
(E x R06) + (E x R6) = R06 (multiply out)
R06 + R6 = R06/E (divide both sides by E)
R6 = (R06/E) - R06 (minus R06)
Substitute the numbers:
R6 = (60/0.75)-60 = 20 Angstrom.
Does this make sense? The two probes must be closer than 60 A to get a transfer efficiency above 50%.
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You are an absolute star thankyou so much!!!
Yes it makes much more sense now I understand what they mean!! [:o)] [:)] [:)] I can get on with it rather than staring blankly at the page!!! I dont usuall like posting work related things up here but google was no help...unless I wanted to know the FRET spacing on a guitar!!! [;D]
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I'm a bit (OK, a lot) rusty, but it makes sense to me. Don't blame me if I've missed something though please!! [;D]
Actually brings back how interesting I found all this. FRAP especially was lovely to do - fluorescence recovery after photobleaching. Basically we used the confocal to bleach a tiny patch of fluorescence in a cell, then calculated how long it took the fluorescence to recover as a measure of how mobile the fluorescently-labelled proteins in the cell were. Got the paper published in JBC. (O'Toole et al, JBC 2003; 278(46):45770-6 if you're interested)
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Oh yes, found a couple of web sites (got the graph from the second one):
http://en.wikipedia.org/wiki/Resonance_energy_transfer (http://en.wikipedia.org/wiki/Resonance_energy_transfer)
http://www.anatomy.usyd.edu.au/mru/fret/abot.html#rzero (http://www.anatomy.usyd.edu.au/mru/fret/abot.html#rzero)