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ChIP assays were performed using human U2OS cells. A titration consisting of 1, 5 and 10 ul of antibody per ChIP experiment was analyzed. IgG (1 ug/ IP) was used as a negative IP control. Quantitative PCR was performed with primers for the promoter of the constitutively expressed GAPDH gene and for myoglobin exon 2. 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 trimethylation of K4 at histone H3 is associated with the promoters of active genes.

Western Blot (Cell lysate) analysis of 15 ug histone extracts of HeLa cells.

ELISA is a quantitative method used to determine the titer of the antibody using a serial dilution of antibody against human Histone H3 (K4me3) in antigen coated wells. 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 1:19000.

Cross reactivity tests using the Histone H3 (K4me3) antibody.
Dot Blot analysis was performed with peptides containing other H3K4 methylations and the unmodified sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20000. The figure shows a high specificity of the antibody for the modification of interest.