H4K5 8 12 16ac Polyclonal Antibody

To test the cross-reactivity of the Bioss antibody against H4K5,8,12,16ac (cat. No. bs-53098R), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. The figure shows a high specificity of the antibody for the modification of interest.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H4K5,8,12,16ac (cat. No. bs-53098R) 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 1:21,200.

ChIP was performed with 2 \u03bcg of the Bioss antibody against H4K5,8,12,16ac (cat. No. bs-53098R) on sheared chromatin from 1 million HeLa cells using a ChIP-seq kit as described above. The IP\u2019d DNA was subsequently analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer\u2019s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure shows the signal distribution along the complete length of chromosome 5 (figure A) and a zoomin to a 600 kb region (figure B). Figure C and D show the enrichment in two genomic regions on chromosome 3 and 12, respectively, containing EIF4A2 and GAPDH positive controls. The position of the amplicon used for validating the QPCR results is shown with an arrow.

ChIP assays were performed using human HeLa cells, the Bioss antibody against H4K5,8,12,16ac (cat. No. bs-53098R) and optimized PCR primer sets for qPCR. ChIP was performed with a ChIP- seq kit on sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5 and 10 \u03bcg per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active gene EIF4A2 and for a region 1 kb upstream of the GAPDH gene, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat region used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).