I´m trying to do an Western blot against some histone modification. I´ve tried with just nuclei in my sample and also after extracting histone. The problem is: my antibodies detect band in places that should be no bands. For example: my H1k26Ac detect bands not in the middle of the membrane (where it should be moreless) but at the bottom just like it was an H4.
I also am stripping the membrane with the acid protocol and then after re-probing with h3 (my marker) it seems that the gel was totally wrong loaded (however I had quantified it before).
So my questions are:
1) It is really necessary to do histone extraction for this thecnique?!
2) monoclonal antibodies could be "wrong" on their target?
3) the acid stripping protocol is better or worse thet the other with beta-mercaptoethanol for histone modifications.
4) Can I stripp more than once the same membrane and yet have good results?
Detecting histone modifications
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