Re: FRET results
Posted by
Zoltan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FRET-results-tp2580297p2580864.html
Hi,
We use 514 nm for yfp and 405 or 458 for cfp. Please be aware of the potential artifact that comes from bleaching ypf and then exciting cfp with 405:
We do get a very strong (artificial) signal in the donor channel when using 405 nm even in a control prep where there is no donor at all! It seems to be a conversion of the yfp molecules to a form that can be easily excited by 405, but not nearly as much by 458. There was a paper about this around 2000 in Nature, I think. Some authors say they don't see this conversion, but we do. Our recipe now is to excite cfp with 458.
I hope this helps,
Zoltan
On Fri, Apr 3, 2009 at 12:55 PM, ramesh kandpal
<[hidden email]> wrote:
hi,
can anyone give some idea that which is the most appropriate laser wavelength for performing acceptor photobleaching method of CFP/YFP FRET. If 488nm laser is used for YFP excitation is there any possibility of loosing FRET data. What difference does it create in FRET results if we use 514nm laser line.
thanks & best wishes,
RK
--
Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK