Re: FRET on Alexa Stained Antibodies

Dear Michael,

I have not use this experimental setup, because we do not have the 514 
excitation option (instead we have 532 nm, which is better). However this 
combination might work, although the 514 excitation is not optimal for 
Alexa 555. Both Alexa 546 and 555 have a shoulder around 514 nm, so they 
can be excited there.  Alexa 546 is more sensitive to bleaching than Alexa 
555, in addition Alexa 555 have higher absorption coefficient.	So probably 
Alexa 555 is a better choice.

But unfortunately you should do the experiments to see how it works!

Good luck

Janos


At 02:55 PM 4/24/2007, you wrote:

>Hello All,
>	  A researcher in our institute would like to observe FRET in the 
> following way.
>	  I want to observe FRET between conjugated antibodies. The donor 
> fluorochrome being Alexa 555 and the acceptor fluorochrome Alexa 647. 
> Therefore would need to excite the Alexa 555 and observe emission from 
> the Alexa 647.
>
>We have a FACSAria and a FACSVantage with the possibility of 514 
>excitation. Has anyone done anything similar or can you help as to how 
>this can be achieved.
>
>Mike Hughes, Flow Cytometry, PICR, Manchester, UK
>
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Janos Szollosi
Department of Biophysics and Cell Biology
Faculty of Medicine
Medical and Health Science Center
University of Debrecen
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