Polyclonal antibody raised in rabbit against histone H3 acetylated at lysines 9 and 14 (H3K9/14ac), using a KLH–conjugated synthetic peptide.

ChIP results obtained with the antibody directed against H3K9/14ac
ChIP assays were performed using HeLa cells, the antibody against H3K9/14ac (Cat. No. 25264) and optimized primer pairs for qPCR. ChIP was performed using sheared chromatin from 1.5 million cells. A titration of the antibody consisting of 1, 2, 5 and 10 μg per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. QPCR was performed using primers specific for the promoter of the ACTB gene as a positive control target and for exon 2 of the MB gene as a negative control target. Figure 1 shows the recovery (the relative amount of immunoprecipitated DNA compared to input DNA). These results confirm the observation that acetylation of H3K9/14 is present at active promoters.

ChIP-seq results obtained with the antibody directed against H3K9/14ac
ChIP was performed with 1 μg of the antibody against H3K9/14ac (Cat. No. 25264) on sheared chromatin from 1 million HeLaS3 cells. IgG (2 μg/IP) was used as a negative IP control. The immunoprecipitated DNA was analysed by QPCR with optimized PCR primer pairs for the promoters of the active GAPDH and c-fos genes, used as positive control targets, and the coding region of the inactive MB gene and the Sat2 satellite repeat, used as negative control targets (figure 2A). The immunoprecipitated DNA was subsequently analysed with an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2 shows the peak distribution along the complete sequence and a 800 kb region of the X-chromosome (figure 2B and C) and in 100 kb regions surrounding the RBM3, GAPDH and c-fos genes (figure 2D, E and F). These results clearly show an enrichment of the H3K9/14 double acetylation at the promoters of active genes.

Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against H3K9/14ac (Cat. No. 25264), 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 (Figure 3), the titer of the purified antibody was estimated to be 1:5,900.

Cross reactivity tests using the antibody directed against H3K9/14ac
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K9/14ac (Cat. No. 25264) with peptides containing other histone modifications and the unmodified H3K9/14 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H3K9/14ac
Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the antibody directed against H3K9/14ac (Cat. No. 25264) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Immunofluorescence using the antibody directed against H3K9/14ac
Mouse NIH3T3 cells were stained with the antibody against H3K9/14ac (Cat. No. 25264) and with DAPI. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K9/14ac antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

50 µg/36 µl

PBS containing 0.05% azide and 0.05% ProClin 300

Human, mouse

Affinity purified

synthetic peptide

Aqueous buffer solution

Store at –80°C for up to 2 years. Centrifuge after first thaw to maximize product recovery. Aliquot to avoid repeated freeze/thaw cycles. Aliquots may be stored at –20°C for at least one month.

ChIP/ChIP – seq (1 – 2 µg/IP)
ELISA (1:100)
DB (1:20,000)
WB (1:1000)
IF (1:500)

The optimal antibody amount per IP should be determined by the end–user. We recommend testing 1–5 µg per IP

Avoid freeze/thaw cycles

Antibodies

Antibody