Thinking about doing FRET - please point me in the right direction
ian.clements at probes.com
Thu Jan 25 13:29:23 EST 2001
There is a great deal of information out there on FRET and its not easy
to pick a starting point. A good book that has probably more
information than you need is "Fluorescence Resonance Energy Transfer:
Theory and Data" 1994 sorry I can't remember the publisher or authors, I
think the first one is V.W. Van der Meer. This book has about the most
extensive collection of data on donor-acceptor pairs.
In part what you choose will be dictated by your instrumentation so take
into consideration what you have at your disposal. If you have a fairly
decent spectroflorimeter then you are pretty much in business. Then
you'll have to figure out which chemistry you are going to use to attach
the dyes, not all options are available in every reactive form.
There are a couple of different approaches to FRET. You could go the
dye-quencher route and look for loss of signal upon association. Pretty
much any dye used with DABCYL as the quencher seems to work.
Fluorescein (FITC) and rhodamine (TRITC) are probably the two most
widely used FRET pair for fluorescence emission FRET.
Dima Klenchin wrote:
> I am thinking that a protein I study might self associate
> under some specific conditions (binding to cell organells).
> I have a lot of the protein available and I was thinking if
> doing FRET is a good way to assay for this kind of thing.
> The idea is to covalently label two pools of protein with two
> different dyes, donor and acceptor, and then see if and when
> there is an increased fluorescence appearing.
> Problem is, I don't know much about the technique and thus
> far can hardly make an educated choice of what dyes to use,
> nor I can sense any caveats in using this approach.
> I just started to dig through the literature and so far results
> are chaotic.
> Any comments will be greatly apprecaited. Is this feasible
> at all? Where to start? Specific books, papers I must read?
> Any other advice?
> Thank you.
> - Dima
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