Unintended fret

Randy T. Fischer fischer1 at mail.nih.gov
Mon Jun 5 17:08:27 EDT 2006


Ralph,

Many years ago we saw this very same problem, using slightly different
fluorophores (PE and PE-Cy5) using antibodies to two parts of the same
surface molecule (anti-idiotype and anti-VH antibodies).  Once seen easily
corrected by putting the antibodies into channels that did not have this
potential for FRET.  At the time, we considered it a problem, never dreaming
it would become such a powerful scientific tool.

Randy T. Fischer, NIH/NIAMS
B Cell Biology Group
9000 Rockville Pike
Bldg 10, Room 6D50
Bethesda, MD 20892
(301) 594-3537 (voice)
(301) 402-2209 (fax)



From: Rossi Ralph <ralph.rossi at petermac.org>
Date: Thu, 1 Jun 2006 17:14:45 +1000
To: cyto-inbox
Conversation: Unintended fret
Subject: Unintended fret

Dear all, I have some users who use the combination of APC and APCCY7 , it
seems to me that 
there's an issue of unintentional Fret occuring.As reported by the
investigators  " the staining level
is less( or barely visible) in the sample than with all the fluophores
combined" than the single colour controls.
I would assume if the epitopes are close enough this can certainly happen ,
as in "intended" fret. Has anyone
had this experience with phenotype samples ?

thanks 



regards 
		  ralph

Ralph Rossi 
Flow facility Manager
Peter MacCallum Cancer Institute
Melbourne, Australia
email  r.rossi at pmci.unimelb.edu.au





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