Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against H4K5, 8, 12ac in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:14, 500.

HeLa cells were fixed with 4% formaldehyde and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. Image shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. B, C, D and E: staining of the cells with the H4K5, 8, 12ac antibody after incubation of the antibody with 10 ng/ul of the following blocking peptides: H4K5, 8, 12 unmodified (B), H4K5, 8, 12ac (C), H2A.ZK5, 7, 11ac (D) and H4K5, 8, 12, 16ac (E).

A Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4 shows a high specificity of the antibody for the modification of interest.

ChIP was performed with the "iDeal ChIP experiment was analysed. IgG (1 ug/IP) was used as negative IP control. QPCR was performed with primers for promoter of the active gene EIF4A2 and for a region 1 kb upstream of the GAPDH gene, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat region used as negative controls. Image shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).