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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