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Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H3K4un ChIP assays were performed using HeLa cells, the monoclonal antibody against H3K4un (Cat. No. MAb-149-050) and optimized PCR primer sets for qPCR. Chromatin was sheared with the Diagenode Bioruptor. ChIP was performed with the “LowCell# ChIP” kit (Cat. No. kch-maglow-016), using sheared chromatin from 105 cells. A titration of the antibody consisting of 1, 5 and 10 μg per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the GAPDH promoter (Cat. No. pp-1001-050) and for exon 2 of the myoglobin gene (Cat. No. pp-1006-050). 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. Cross reactivity of the Diagenode monoclonal antibody directed against H3K4un To test the specificity an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody against H3K4un (Cat. No. MAb-149-050). The wells were coated with peptides containing the unmodified H3K4 region as well as the mono-, di- and trimethylated H3K4. Figure 2 shows a high specificity of the antibody for the unmodified peptide.
Figure 3. Western blot analysis using the Diagenode monoclonal antibody directed against H3K4un Histone extracts (15 μg) from HeLa cells were analysed by Western blot using the Diagenode monoclonal antibody against H3K4un (Cat. No. MAb-149-050) 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 4. Immunofluorescence using the Diagenode monoclonal antibody directed against H3K4un HeLa cells were stained with the Diagenode antibody against H3K4un (Cat. No. MAb-149-050) 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 H3K4un antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. 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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