Howard (as usual) is probably right. 7-AAD absorbs out past 600 nm and its absorption strongly overlaps the emission of R-PE. Absorption and fluorescence emission spectra of 7-aminoactinomycin D (A-1310) bound to DNA. [Spectra: 12KB] Absorption and fluorescence emission spectra of R-phycoerythrin (P-801) in pH 7.5 buffer. [Spectra: 12KB] Howard Shapiro wrote: > Brian Newsom wrote- > > >Question to all of you DNA experts. We have someone who is tring to run an > >experiment staining a nuclear localized protien with PE and then staining > >with 7-AAD. The 7-AAD staining works well with a good CV. The antibody > >staining looks decent (although low percentage and fairly dim) when by > >itself, but when the 7-AAD is added the antibody staining totally goes away. > >Is there an energy transfer or steric hinderance issue that may be going on? > > Steric hindrance is unlikely to be the problem since the antibody gets in > in the absence of 7-AAD, which itself is not that big a molecule. The loss > of PE fluorescence is almost certainly due to energy transfer to the > 7-AAD. Since energy transfer works at very short range, we don't expect or > see this effect when surface antigens are stained with PE- (or > fluorescein-) labeled antibodies and 7-AAD (or PI) is used to stain nuclei, > but it can be very noticeable when working with nuclear antigens, as is the > case here. > > -Howard
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