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When staining transciption factor proteins with fluorescent antibodies (Alexa, etc.), why are the fluorescence signals stronger in the nucleus as compared to fluorescence signals in the cytoplasm (in context of transcription factor-activation?) Is it simply spatial localization, that more fluoresence-marked proteins are at the same place, or are there other factors related?

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    $\begingroup$ Can you please clarify what you want to know. —— Do you find a strong signal in the nucleus surprising? Given their function, where would you expect to find transcription factors? $\endgroup$
    – tyersome
    Dec 10, 2019 at 18:30

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Yes, your hypothesis is correct. There are more of them.

A few terms need clarifying here, just in case...

First, fluorescence is due to the property of a dye that can be conjugated to the antibody. Different fluorophores have different properties, some are more efficient than others. Some display strong "solvatochromism", i.e. they are happier in the hydrophobic environment (brighter and red shifted).

Second, DNA is stained not with an antibody but with the UV fluorophore DAPI, which shines brighter in DNA —same thing with ethidium bromide and Sybr Safe.

Now, if you are labelling a TF and they ought to be in the nucleus you definitely will have a brighter signal as you suggest. There will be a way-ever-so slight difference as the environment is less aqueous, but the nucleus is not that packed as on pictures.

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