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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K9me1 ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against H3K9me1 (cat. No. CS-065-100) and optimized PCR primer sets for qPCR. Chromatin was sheared with the Diagenode “Shearing ChIP” kit (cat. No. kch-redmod-100). ChIP was performed with the “OneDay ChIP” kit (cat. No. kch-oneDIP-060), using sheared chromatin from 1.6 million cells. A titration of the antibody consisting of 2, 5, 10 and 15 μl per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. Quantitative PCR was performed using primers for the promoter of the housekeeping gene GAPDH and for the coding region of the myogenic differentiation gene (MYOD), a gene that is inactive at normal conditions. 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 H3K9me1 (cat. No. CS-065-100). 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 1:50,000.
Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K9me1 A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K9me1 (cat. No. CS-065-100) with peptides containing other modifications of histone H3. Other histone modifications include di- and trimethylation of the same lysine and mono-, di- and trimethylation of lysine 27, 36 and 79. 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 1:200,000. 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 H3K9me1 Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K9me1 (cat. No. CS-065-100) diluted 1:1000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right.
Figure 5. Immunofluorescence using the Diagenode antibody directed against H3K9me1 HeLa cells were stained with the Diagenode antibody against H3K9me1 (cat. No. CS-065-100) and with DAPI. Cells were formaldehyde fixated, permeabilized with Triton X-100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K9me1 antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight. Figure 5B: staining of the nuclei with DAPI, which specifically labels DNA. Both antibody and DAPI staining are restricted to the nucleus.
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