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ChIP assays were performed using human HeLa 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 IP control. QPCR was performed with primers for the promoter of the active gene GAPDH as a negative control, and for the coding regions of the inactive genes MYT1 and TSH2B as positive controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H3K27me3S28p is preferably present at inactive genes.

ChIP was performed on sheared chromatin from 1.5 million colcemid treated HeLaS3 cells using antibody. The figure shows the enrichment in genomic regions surrounding the TSH2B and MYT1 positive control genes, and of chromosome 3 and 20.

Western Blot (Cell lysate) analysis of (1) 15 ug histone extracts of Hela cells, (2) 1 ug of recombinant histone H3.

Western Blot analysis of different stages of the cell cycle (1) asynchronous population, (2)G1/Sa, (3)G1/Sb, (4)S phase, (5)G2/M, (6)M, (7)G1. The figure shows a nice peak of H3K27me3S28p expression during mitosis.

ELISA is a quantitative method used to determine the titer of the antibody using a serial dilution of antibody against Histone H3 (K27me3)(phospho S28). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:4300.

Cross reactivity test using the Histone H3 (K27me3)(phospho S28) antibody.
Dot Blot analysis was performed with peptides containing other modifications of histone H3 and H4 and the unmodified H3K27S28 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2000. The figure shows a high specificity of the antibody for the modification of interest.