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ChIP assays were performed using human HeLa cells. A titration consisting of 1, 2, 5 and 10 ug of antibody per ChIP experiment was analyzed. IgG (2 ug/IP) was used as a negative IP control. QPCR was performed using primers specific for the promoters of the ACTB and EIF4A2 genes, used as positive control targets and for the coding region of the MYT1 gene, used as a negative control target. The figure shows the recovery (the relative amount of immunoprecipitated DNA compared to input DNA). These results confirm the observation that acetylation of Histone H2AZ is present at active promoters.

ChIP was performed on sheared chromatin from 1 million HeLaS3 cells. IgG (2 ug/IP) was used as a negative IP control. The figure shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome and in 100 kb regions surrounding the EIF4A2, ACTB and GAPDH genes. These results clearly show an enrichment of the Histone H2AZ acetylation at the promoters of active genes.

Immunofluorescent staining of Hela cell line with antibody followed by an anti-rabbit antibody conjugated to Alexa488 (left). The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings (right).

ELISA is a quantitative method used to determine the titer of the antibody using a serial dilution of antibody against Histone H2AZ, crude serum and flow through 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 1:8800.

Cross reactivity test using the Histone H2AZ antibody.
Dot Blot analysis was performed with peptides containing other histone acetylations and the unmodified Histone HAZ 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.