H4K8ac Antibody

Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K8ac
ChIP assays were performed using human HeLa cells, the Diagenode antibody directed against H4K8ac (cat. No. pAb-103- 050) and optimized PCR primer sets for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016) on sheared chromatin from 10,000 cells using the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5, and 10 μg per ChIP experiment was analysed. IgG (1 μg/IP) was used as negative IP control. QPCR was performed with primers for the c-fos promoter and for the Sat2 satellite repeat region. 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 antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K8ac (cat. No. pAb-103-050), crude serum and flow through in antigen coated wells. 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 purified antibody was estimated to be 1:16,700.

Figure 3. 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. pAb-103-050) with peptides containing other histone modifications and the unmodified H4K8. One hundred to 0.2 pmol of the respective peptides 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 4. Western blot analysis using the Diagenode antibody directed against H4K8ac
Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody directed against H4K8ac (cat. No. pAb-103-050) diluted 1:200 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 5. Immunofluorescence using the Diagenode antibody directed against H4K8ac
Mouse NIH3T3 cells were stained with the Diagenode antibody against H4K8ac (cat. No. pAb-103-050) 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 H4K8ac antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.