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Figure 1. ChIP results obtained with the Diagenode antibody directed against H2AK5ac ChIP assays were performed using human HeLa cells, the Diagenode antibody against H2AK5ac (Cat. No. C15410215) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. C01010051), using sheared chromatin from 1.5 million 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 a region approximately 1 kb upstream of the GAPDH and ACTB promoters, used as positive controls, and for the coding region of the inactive MYOD1 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).
Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H2AK5ac ChIP was performed on sheared chromatin from 1.5 million HeLaS3 cells using 0.5 μg of the Diagenode antibody against H2AK5ac (Cat. No. C15410215) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the enrichment along the complete sequence and a 1 Mb region of the X-chromosome (fig 2A and B) and in genomic regions of chromosome 7, surrounding the ACTB gene, and of chromosome 12, surrounding the GAPDH gene (fig 2C and D). The position of the amplicon used for ChIP-qPCR is indicated by an arrow.
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 H2AK5ac (Cat. No. 15410215) 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 2), the titer of the antibody was estimated to be 1:25,000.
Figure 4. Cross reactivity tests using the Diagenode antibody directed against H2AK5ac To test the cross reactivity of the Diagenode antibody against H2AK5ac (Cat. No. 15410215), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H2AK5. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:5,000. Figure 3 shows a high specificity of the antibody for the modification of interest.
Figure 5. Western blot analysis using the Diagenode antibody directed against H2AK5ac 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 H2AK5ac (Cat. No. C15410215). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left
Figure 6. Immunofluorescence using the Diagenode antibody directed against H2AK5ac HeLa cells were stained with the Diagenode antibody against H2AK5ac (cat. C15410215) 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 H2AK5ac 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.
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