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Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K12ac ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K12ac (Cat. No. C15410331) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010055) on sheared chromatin from 1,000,000 cells. A titration of the antibody consisting of 0.5, 1, 2 and 5 μg per ChIP experiment was analysed. IgG (1 μg/IP) was used as negative IP control. QPCR was performed with primers for promoter of the active GAPDH and EIF4A2 genes, 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).
Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K12ac ChIP was performed with 0.5 μg of the Diagenode antibody against H4K12ac (Cat. No. C15410331) on sheared chromatin from 1,000,000 HeLa cells using the “iDeal ChIP-seq” kit as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq 2000. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the peak distribution along the complete sequence and a 2 Mb region of the human X chromosome (figure 2A and B) and in two genomic regions surrounding the GAPDH and EIF4A2 positive control genes (figure 2C and D).
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 H4K12ac (Cat. No. C15410331) 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 antibody was estimated to be 1:14,300.
Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K12ac To test the cross reactivity of the Diagenode antibody against H4K12ac (Cat. No. C15410331), 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 1:1,000. Figure 4 shows the antibody is specific for the K12 acetylation with some slight cross reaction with K8ac.
Figure 5. Western blot analysis using the Diagenode antibody directed against H4K12ac Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K12ac (Cat. No. C15410331). The antibody was diluted 1:500 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 H4K12ac HeLa cells were stained with the Diagenode antibody against H4K12ac (Cat. No. C15410331) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K12ac antibody (left) diluted 1:200 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.
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