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Detection of Histone H3 Ser10 phosphorylation by Western blot


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#1 Faust

Faust

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Posted 02 November 2010 - 08:18 AM

I have some problems to get nice blots of phospho Histone H3 (Milipore antibody). Usually I use this extraction protocol with works quite nice and I get a good yield of Histones which I can see by staining the blot with total Histone H3 antibody.

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My problem is that the phosphorylation state does not really make sense and is not reproduceable. Additionally I add NaF and Na3VO4 in the TEB Buffer to avoid dephosphorylation. Is the acid interfering with the activity of these inhibitors?
Does anybody know a nice protocol which allows me to detect this phosphorylation by western blot?

Thanks a lot!




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