Re: FRET on Alexa Stained Antibodies

Hi Mike,

It would seem from your cut and paste that the people have already bought the stuff so take Janos's advice and suck it and see (though it's very much like the cy3/cy5 pairing and should work fine).  If it doesn't work, get them to consult you during their planning stage (I refrained from using quotes around "planning", but not for long).  

You don't really go into much detail about what lines you have available other than 514 (I'm guessing you've got a 633, 635 or 647 in there too though, so you can measure the acceptor fluorescence), but there might be some better combination for your set-up, and it would be better for all of you if your punters found out from you which dyes you could excite, before they worked out which ones they'd impose on you, along the lines of...

"I want to observe FRET between conjugated antibodies. Which donor and acceptor fluorochrome will work best on your Vantage or Aria ?"


Cheers

Ray





----- Original Message ----- 
  From: Janos, Szollosi 
  To: Cytometry Mailing List 
  Sent: Wednesday, April 25, 2007 10:51 AM
  Subject: 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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