Hello, I am treating SH-SY5Y cells with different drugs of which cause Parkinson. during western blot i see that acetylation of H3 or H4 increase decrease which is fine but why my total level of H3 and H4 show increase or decrease. it should be same for all treated cells.Your opinion will be highly appreciated.
From a western blot stand point, does your total H3/H4 signal decrease when your acetylated H3/H4 increases and vice versa? If so, it has to do with the incomplete stripping. I have seen this before when measuring phosphorylated protein levels by Western. If the protein is non-acetylated, it does not bind to the acetylated antibody and binds better to the total antibody. If it is acetylated and binds to the acetylated antibody, it doesn’t seem to bind as much to the total antibody even if you strip the membrane in between.
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