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Dear Confocalists,

I would greatly appreciate your advice on the topic I don't know much about:
I am imaging several proteins belonging to a Hox group of 
transcription factors, which regulate multiple (hundreds) of genes by 
specifically binding DNA on single and clustered sites. I visualize 
these proteins using indirect immunofluorescence (primary polyclonal 
and secondary antibodies fused to Alexa dyes) in fixed Drosophila 
embryos and image them on Leica SP2 confocal microscope At high 
magnification, 63X and higher, these transcription factors appear as 
foci, or speckles, within the nucleus. I observe this speckled 
localization with a variety of antibodies against several 
transcription factors. After deconvolution using Autoquant software, 
these foci become even more profound, about 60 of them per nucleus.
Now here are my questions:
Are these foci real or an artifact (of fixation, for example)?

If they are real, are they just random clusters of proteins inside 
the nucleus or do I actually observe clusters of transcription 
factors bound to their DNA target sequences?

Does anybody have an estimate of how many proteins need to be 
clustered together in order to be detected with polyclonal 
antibodies/secondary antibodies with 5-6 fluorophores each, in fixed 
tissue (about 10-20 microns under the coverslip)?

I realize that some of these questions will probably not have general 
answers ( that is, the answers will vary depending which 
transcription factor and which system one is working with), but any 
input from people who image transcription factors or any referrals to 
the relevant literature will be greatly appreciated. 

Best wishes,

Ella Tour

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Ella Tour, Ph.D.
Department of Cell and Developmental Biology, 0349
University of California, San Diego
9500 Gilman Drive, 4305 Bonner Hall
La Jolla, CA  92093-0349
Phone 858-822-0461
FAX 858-822-0460
email: [log in to unmask]