I am trying to look at acetylated histone levels and the problem is that my Western blot results are showing increasing levels of both acetylated and total histone levels in Compound X treated cells compared to control cells.
When i look at beta-actin or GAPDH the levels are same.
Using RIPA buffer got making whole cell lysate
In my opinion, it is hard to extract all the histones when you using RIPA or other common lysis buffer to got the whole cell lysates, it is likely dependent on the chromatin status. For example, when your treat the cells with DNA damage reagents, the chromatin become loose and you may get more total histones using RIPA buffer.
So I recommend you to use acid extraction method to got the histones.
Good luck
http://www.nature.com/nprot/journal/v2/n6/full/nprot.2007.202.html#B24
http://www.abcam.com/ps/pdf/protocols/Histone%20extraction%20protocol.pdf
I agree about acid extraction. Also, keep in mind that it is possible that your total amount of histone is really increasing in the treated cells, if they increase histone expression or decrease histone degradation... See if your cells look healthy after treatment, and if they look really unhealthy then it is possible that they may have many phenotypes.
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