Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against H3K27me1, crude serum and flow through. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 1), the titer of the purified antibody was estimated to be 1:32, 900.

Human U2OS cells were fixed with 4% formaldehyde for 20' and blocked with PBS/TX-100 containing 5% normal goat serum. A: cells were labeled with the H3K27me1 antibody (left) at 1:1,000 followed by an anti-rabbit conjugated to Alexa568 or with DAPI (right). B, C, D and E: staining of the cells with the H3K27me1 antibody after incubation of the antibody with 2 ng/ul blocking peptide containing the unmodified and the mono-, di- and trimethylated H3K27, respectively.

A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K27me1 with peptides containing other modifications and unmodified sequences of histone H3 and H4. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. 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 using HeLa cells: ChIP” kit, using sheared chromatin from 100,000 cells. A titration of the antibody consisting of 1, 2, 5 and 10 ug per ChIP experiment was analysed. IgG (2 ug/IP) was used as negative control. qPCR primers were for the promoter and the coding region of the active gene GAPDH negative and a positive control target, respectively. Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR analysis).