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Figure 1 ChIP results obtained with the Diagenode monoclonal antibody directed against H4K20me3 ChIP assays were performed using human HeLa cells, the Diagenode monclonal antibody against H4K20me3 (cat. No. SN-148-100) and optimized PCR primer sets for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. Two different quantities of antibody (3 and 12 μl per ChIP experiment) were analysed. IgG (1 μg/IP) was used as negative IP control. QPCR was performed with primers for the GAPDH promoter and for the inactive gene TSH2B. 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 Western blot analysis using the Diagenode monoclonal antibody directed against H4K20me3 Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode monoclonal antibody against H4K20me3 (cat. No. SN-148-100) diluted 1:50 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 3 Immunofluorescence with the Diagenode monoclonal antibody directed against H4K20me3 HEK293T cells were stained with the Diagenode antibody against H4K20me3 (cat. No. SN-148-100, green), with human autoantibodies against the centromeres (CREST, Cortex Biochem, pink) and with DAPI (blue). Cells were fixed with 4% paraformaldehyde and blocked with PBS containing 0.1% Tween, 2% BSA, and 5% normal goat serum. Cells were immunofluorescently labeled with the H4K20me3 antibody (diluted 1:200 in blocking solution) and the CREST antibody (diluted 1:1000 in blocking solution) by overnight incubation at 4°C. Subsequently cells were labeled with goat-anti-mouse antibody conjugated to Alexa488 and goat-anti-human antibody conjugated to Alexa633 (both diluted 1:200 in blocking solution and incubated for 1 hour at RT). Cells were mounted with Vectashield containing DAPI. Immunofluorescence wwas performed by Dr. E. Baart, Erasmus University, Rotterdam, the Netherlands.
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