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ChIP assays were performed using human HeLa cells. A titration of the antibody consisting of 0.5, 1, 2 and, 5 ug per ChIP experiment was analysed. IgG (1 ug/IP) was used as negative IP control. QPCR was performed with primers for a region approximately 1 kb upstream of the GAPDH promoter and for the EIF4A2 promoter, used as positive controls, and for the coding region of the inactive MYOD1 gene and the Sat2 satellite repeat, used as negative controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

ChIP was performed on sheared chromatin from 1.5 million HeLaS3 cells using 5 ug of antibody. The figure shows the enrichment along the complete sequence and a 1 Mb region of the X-chromosome and in genomic regions of chromosome 12 and 3, surrounding the GAPDH and EIF4A2 positive control genes.

ELISA is a quantitative method used to determine the titer of the antibody using a serial dilution of antibody against Histone H3 (K56ac) 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 1:15300.

Cross reactivity tests using the Histone H3 (K56ac) antibody.
Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K56. 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.