Determination of the titer To determine the titer, an ELISA was performed using a serial dilution of the antibody against H4K20me1. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,000.

Dot Blot was performed with peptides containing other modifications of histone H4 or the unmodified sequence. Other histone modifications include mono- and dimethylation of the same lysine and acetylation of the nearby lysine 16. To determine the cross reactivity, 0.2 to 100 pmol of peptides were spotted on a membrane. Three different peptides for H4K16ac were used. The antibody was used at a dilution of 1:20,000. Image shows a high specificity of the antibody for the modification of interest.

ChIP assays were performed using human U2OS cells. IgG (5 ug/IP) was used as negative control. qPCR primers were for the GAPDH promoter and for the coding region of MYOD, a gene that is inactive at normal conditions. Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR analysis).