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Figure 1. Determination of the antibody titer To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human H4K8ac (cat. No. CS-103-100). 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 1:17,500.
Figure 2. Cross reactivity test using the Diagenode antibody directed against H4K8ac A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H4K8ac (cat. No. CS-103-100) with peptides containing other modifications of histone H4 and H3 and an unmodified histone H4 sequence. One hundred to 0.2 pmol of the 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 3. Western blot analysis using the Diagenode crude serum against H4K8ac Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against JMJD2c (Cat. No. CS-105-100) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest (expected size: 120 kDa) is indicated on the right; the marker (in kDa) is shown on the left. The smaller fragment of approximately 92 kDa may represent a splicing variant.
Figure 4. ChIP results obtained with the Diagenode crude serum directed against H4K8ac Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H4K8ac (cat. No. CS-103-100) diluted 1:250 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. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-103-050) for 1 hour at room temperature.
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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