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Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me1 ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me1 (cat. No. C15410034) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. AB-001-0024) on 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. 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. Determination of the titer To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me1 (cat. No. C15410034) 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 1:51,100.
Figure 3. Cross reactivity tests using the Diagenode antibody directed against H4K20me1 To check the specificity of the Diagenode antibody against H4K20me1 (cat. No C15410034) 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 antibody directed against H4K20me1 Western blot was performed on whole cell extracts (25 μg, lane 1) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 2, 3, 4 and 5, respectively) using the Diagenode antibody against H4K20me1 (cat. No. C15410034). 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 antibody directed against H4K20me1 HeLa cells were stained with the Diagenode antibody against H4K20me1 (Cat. C15410034) 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 H4K20me1 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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