This is a nice post. FRET (and FLIM) are great techniques; I'd quibble with one thing you say: "FRET is less complicated than it sounds."

The procedure is fairly straightforward. It's just a process of replacing pieces of protein with fluorescent protein, etc. as you say.

Here's a couple of rubs, however:

1. The signal-to-noise ratio is very subtle. One has to weed through a lot of raw data to pull out signal. Even under perfect conditions.

2. While one can extrapolate the distances between the two fluors via the difference in excitation, without any way to triangulate the signal, it is impossible to determine the directional motion of the fluor. You just know that the distance has changed linearly. Could be rotational, angular, twisting, etc. but you just can't know.

You'll notice that most papers on FRET will remark (if they're honest) that they needed to try 6 or 7 protocols to get their results. This, as you can imagine, is frustrating.

We do FRET in my lab. The front end is easy; it's the back end that gives us hives.

Ah yes. Well I've never got my hands on pretty much any advanced fluorescent technique. Probably part of why I'm so enamored of these flashy methods when I read about them. I haven't had the opportunity to get disillusioned.

They definitely had some convoluted mathematics to get the actual FRET signal separate from CFP contamination and all that. I just skipped over it because it's a pain to explain, and I was more concerned with the conclusions.

If you get a chance to check out the pdf, I'd like to know if you think the direction (rule 2) makes a substantive issue in this case. Seems like all they care about here is whether monomers are sticking to each other.

pdf link is weird. no pdf. :(

shoot.. looks like all my pdfs i've posted are that way.. i must be doing it wrong.. i'll get it fixed by tomorrow morning..

in the meantime, you can get any files i've referenced in the files section of the gnxp forum..