ChIP - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 2 μg of ab4729 (blue), and 20 μl of Protein A/G sepharose beads.
No antibody was added to the beads control (yellow).
The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.
Western blot - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were incubated at 37°C for 6 hours with vehicle control (0 μM) and different concentrations of sodium butyrate (ab120948). Increased expression of histone H3 (acetyl K27)(ab4729) in HeLa cells correlates with an increase in sodium butyrate concentration, as described in literature.
Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 2.5 μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab4927 at 1 μg/ml and ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10,000 dilution and visualised using ECL development solution.
All lanes:
Western blot - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 17 kDa
Exposure time: 10s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
IHC image of ab4729 staining Histone H3 (acetyl K27) in human colon formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer pH 6 for 20 minutes. The section was then incubated with ab4729, 5 μg/ml, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
No primary antibody was used in the negative control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
Western blot - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
All lanes:
Western blot - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 1 µg/mL
All lanes:
HeLa (Human epithelial cell line from cervix adenocarcinoma) histone preparation, nuclear Lysate - Butyrate treated at 2.5 µg
Secondary
All lanes:
Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 17 kDa
Exposure time: 10s
Peptide Array - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)
All batches of ab4729 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - acetyl K27 peptide (ab24404), indicating that this antibody specifically recognises the Histone H3 - acetyl K27 modification.
ab24404 - Histone H3 - acetyl K27
ab15591 - Histone H3 - acetyl K14
ab24003 - Histone H3 - acetyl K18
ab17163 - Histone H3 unmodified
ab48359 - Histone H3 - acetyl K23
ab41409 - Histone H3 - acetyl K36
ab15662 - Histone H4 - acetyl K12
ab16635 - Histone H3 acetyl K9
