Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3 ChIP assays were performed using undifferentiated human teratocarcinoma cells (NCCIT), the Diagenode antibody against H4K20me3 (cat. No. CS-057-100) and optimized PCR primer sets for qPCR. Sheared chromatin from 10,000 cells was used per ChIP experiment. The antibody was tested at two different dilutions of 1:500 and 1:100. The pre-immune serum (PI) was used as a negative control. Quantitative PCR was performed using primer sets for the satellite repeat Sat2 as a positive control and for the promoter of the house keeping gene c-fos, as a negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H4K20me3 is preferably present at heterochromatin.
Figure 2. Determination of the titer To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (cat. No. CS-057-100). 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 1:700.
Figure 3. Cross reactivity test using the Diagenode antibody directed against H4K20me3 Dot Blot was used to check the specificity of the Diagenode antibody against H4K20me3 with peptides containing other modifications of histone H3 and H4. Other histone modifications include monoand dimethylation of the same lysine and mono-, di- and trimethylation of lysines 9, 27 and 36 of H3. 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 1:1,000. 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 H4K20me3 Histone (acid) extracts of NB4 cells were analysed by Western blot using the Diagenode antibody against H4K20me3, diluted 1:750 in TBS-Tween containing 5% skimmed milk. The location of the protein of interest is indicated on the right.
WB Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies.
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