Determination of the titer To determine the titer, an ELISA was performed using a serial dilution of the antibody against H3K36me3. 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 1:12, 700.

Dot Blot analysis was performed to test the cross reactivity of the ab with peptides containing other modifications of histone H3. Other histone modifications include mono- and dimethylation of the same lysine and mono-, di- and trimethylation of lysine 9, 27 and 79. One hundred to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane. The antibody was used at 1:100,000. Image shows a high specificity of the antibody for the modification of interest.

ChIP assays using human U2OS cells. IgG (5 ug/IP) was negative control. qPCR was performed using primer sets for the housekeeping gene GAPDH and for myogenic differentiation gene (MYOD). Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR analysis).

ChIP was performed with 5 ul ab on sheared chromatin from 1 million HeLaS3 cells. IgG (2 ug/IP) was negative control. The IP'd DNA was analysed by qPCR with primers for the coding and promoter region of active GAPDH, for the coding region of inactive TSH2B and for the Sat2 (A). The IP'd DNA was analysed. The 36 bp tags were aligned to the human genome using the ELAND algorithm. B shows the results in 200 kb regions of chromosome 12 (including the GAPDH positive control), 6 and 7 and 14. Results show an enrichment of the H3K36me3 at active genes.