As Howard noted below, energy transfer is likely the cause of diminished nuclear protein labeling in the presence of 7-AAD. (A look at the absorption spectrum of 7-AAD shows that it is excited at the emission range of PE about as efficiently as it is excited by the 488nm laser line.) In addition to a loss of the PE signal, you should see an increase in 7-AAD fluorescence. (If you really want to show 7-AAD is responsible, prelabeling DNA with the non-fluorescent AAD will block 7-AAD binding and should have no effect on PE intensity.) But from a practical standpoint, compatible DNA probes would include DAPI (if a uv laser is available) or TOPRO-3 if a red laser (HeNe, Kr, or diode) is available; PE emission will be unaffected. Moreover, you can still use another 2 or 3 other 488-excited fluorochromes at the same time. Dave ---------------- David M. Coder, Ph.D. Consultant in Cytometry email: d_coder@msn.com tel./messages: 206-499-3446 ----- Original Message ----- From: "Howard Shapiro" <hms@shapirolab.com> To: cyto-inbox Sent: Tuesday, January 29, 2002 4:52 AM Subject: Re: 7-AAD/PE problem > > 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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