Enzyme-linked immunosorbent assay. Read more
Dot blotting Read more
This antibody has been raised in rabbit against histone H3, acetylated at lysine 9 (H3K9ac), using a KLH-conjugated synthetic peptide.
|Species reactivity||Human, mouse, zebrafish, Poplar, P. Falciparum|
|Precautions||This product is for research use only. Not for use in diagnostic or therapeutic procedures.|
|ChIP *||1-2 μg/ChIP||Fig 1, 2|
|Dot Blotting||1:20,000||Fig 4|
|Western Blotting||1:1,000||Fig 5|
* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 μg per IP.
Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K9ac
ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K9ac (Cat. No. C15410004) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. A titration consisting of 1, 2, 5 and 10 μg of antibody per ChIP experiment was analyzed. IgG (2 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers speci c for the promoter of the active genes GAPDH and EIF4A2, used as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat, 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). These results are in accordance with the observation that acetylation of K9 at histone H3 is associated with the promoters of active genes.
Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K9ac
ChIP was performed with 1 μg of the Diagenode antibody against H3K9ac (Cat. No. C15410004) as described above and the IP’d DNA was subsequently analysed on 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 an 800 kb region of the X-chromosome ( gure 2A and B) and in 100 kb regions surrounding the GAPDH and EIF4A2 positive control genes ( gure 2C and D). These results clearly show an enrichment of H3K9ac at the promoters of active genes.
Figure 3. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K9ac (Cat. No. C15410004) in antigen coated wells. The antigen used was a peptide containing the histone modi cation of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:31,700.
Figure 4. Cross reactivity test using the Diagenode antibody directed against H3K9ac
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K9ac (Cat. No. C15410004) with peptides containing other histone modi cations and the unmodi ed H3K9 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 speci city of the antibody for the modi cation of interest. Please note that this antibody recognizes the H3K9 acetylation, both in the presence and the absence of the K14 acetylation.
Figure 5. Western blot analysis using the Diagenode antibody directed against H3K9ac
Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K9ac (Cat. No. C15410004) 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.
Figure 6. Immunofluorescence using the Diagenode antibody directed against H3K9ac
HeLa cells were stained with the Diagenode antibody against H3K9ac (Cat. No. C15410004) and with DAPI. Cells were xed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immuno uorescently labelled with the H3K9ac 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.
|Datasheet H3K9ac C15410004 DATASHEET|
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