H3K27ac polyclonal antibody - Classic
| Name | H3K27ac polyclonal antibody - Classic |
|---|---|
| Supplier | Diagenode |
| Catalog | C15410174 |
| Prices | $295.00 |
| Sizes | 50 μg/42 μl |
| Host | Rabbit |
| Clonality | Polyclonal |
| Applications | ChIPseq ChIP-PCR WB ELISA DB ICC/IF |
| Species Reactivities | Human |
| Purity/Format | Affinity purified |
| Description | Rabbit Polyclonal |
| Supplier Page | Shop |
Product images
Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27acChIP assays were performed using HeLa cells, the Diagenode antibody against H3K27ac (Cat. No. C15410174) and optimized PCR primer sets for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. C01010022), using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5 and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. Quantitative PCR was performed with primers for the promoter of the active ACTB (Cat. No. pp-1005-050) and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and TSH2B genes, 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 H3K27 acetylation is enriched at the pr
Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K27acChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 2 μg of the Diagenode antibody against H3K27ac (Cat. No. C15410174) as described above. Quantitative PCR was performed with primers for the promoter of the active ACTB (Cat. No. pp-1005-050) and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and MB (Cat. No. C17011006) genes, used as negative controls (Figure 2A). 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 2B shows the peak distribution along the complete X-chromosome and in two regions surrounding the ACTB and EIF4A2 positive control genes, respectively (figure 2C and D).
Figure 3. Determination of the antibody titerTo determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27ac (Cat. No. C15410174) 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 1:4,000.
Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27acA Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27ac (Cat. No. C15410174) with peptides containing other histone modifications and the unmodified H3K27 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:25,000. Figure 4 shows a high specificity of the antibody for the modification of interest.
Figure 5. Western blot analysis using the Diagenode antibody directed against H3K27acWestern blot was performed on whole cell (40 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 H3K27ac (Cat. No. C15410174). The antibody was 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 H3K27acHeLa cells were stained with the Diagenode antibody against H3K27ac (Cat. No. C15410174) 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 labelled with the H3K27ac 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.
