Acetylated Histones and Nonhistone Proteins

The first functional cell-based assay for the determination of pan-histone deacetylase activity was achieved by detecting hyperacetylated histones by Western blotting. The histone acetylation level in cells offers a measure of candidate HDACI activity and has been linked to antiproliferative and cytotoxic effects. Beckers et al. used a cellular histone hyperacetylation assay for quantification of the cellular efficacy of HDACIs using the Cellomics Array Scan II platform in 96 wells [14]. The program 

Pan = Total histone deacetylase activities HD( = Individual histone deacetylase isoform activity Numbers 36 and 37 correspond to citation references which report pertinent HDAC isoform activity inhibition of compounds (Cpd) ac-H3 and Ac-H4 represent acetylated histone H3 or H4. 

The total cell extracts were isolated by scraping cells into SDS-lysis buffer. 

Protein concentrations were determined by performing Bradford assay using bovine serum albumin as a standard. Equal amounts 

The separated proteins were transferred to a polyvinylidene difluoride membrane, and nonspecific IgC binding sites were blocked by incubation with 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies. Immune complexes were detected by enhanced chemiluminescence (Amersham Biosciences). 

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