H4K20me1 Antibody

Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H4K20me1
ChIP assays were performed using human HeLa cells, the Diagenode monclonal antibody against H4K20me1 (cat. No. C15200147) and optimized PCR primer sets for qPCR. ChIP was performed with the “the “iDeal ChIP-seq” kit (cat. No. C01010051), using sheared chromatin from 1 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 the coding region of the active GAPDH and ACTB genes, used as positive controls, and for the GAPDH promoter and a region located 1 kb upstream of the GAPDH promoter, 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 monoclonal antibody directed against H4K20me1
ChIP was performed as described above using 0.5 μg of the Diagenode antibody against H4K20me1 (cat. No. C15200147). 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 peak distribution in genomic regions from chromosomes 12, 7, 14 and 3 surrounding the GAPDH, ACTB, FOS and EIF4A2 positive control genes (figure 2A, B, C and D, respectively).

Figure 3. Cross reactivity tests using the Diagenode monoclonal antibody directed against H4K20me1
To check the specificity of the Diagenode monoclonal antibody against H4K20me1 (cat. No C15200147) a Dot Blot was performed with peptides containing different modifications of histone H3 and H4 or the unmodified H4K20 sequence. One hundred to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the modification of interest.

Figure 4. Western blot analysis using the Diagenode monoclonal antibody directed against H4K20me1
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 monoclonal antibody against H4K20me1 (cat. No C15200147). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left, the position of the protein is indicated on the right

Figure 5. Immunofluorescence using the Diagenode monoclonal antibody directed against H4K20me1
Human osteosarcoma (U2OS) cells were stained with the Diagenode antibody against H4K20me1 (cat. No. MAb-147- 100) and with DAPI. Cells were fixed with ice cold methanol for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum. Figure 4A: cells were immunofluorescently labeled with the H4K20me1 antibody (left), diluted 1:200 in blocking solution followed by an anti- mouse antibody conjugated to Alexa488 or with DAPI (right), which specifically labels DNA. Figure 4B: staining of the cells with the H4K20me1 antibody after incubation of the antibody with blocking peptide (cat. No. sp-034-050, concentration: 5 ng/μl).