Recombinant Anti-Histone H3 (acetyl K27) antibody [EP16602] - ChIP Grade (ab177178)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ HeLa cells and 4 µg of Anti-Histone H3 (acetyl K27) antibody [EP16602] - ChIP Grade (ab177178). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

Additional screenshots of mapped reads can be downloaded here.

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 25µg of chromatin, 2µg of ab177178 (blue), and 20µl of Protein A/G Sepharose beads. 2μg of rabbit normal IgG was added to the beads as a control sample (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

Primers and probes are located in the first kb of the transcribed region.

*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) treated (Red)/untreated (Green) with 500ng/ml Trichostatin A for 4 hours with purified ab177178 at 1/1500 dilution. The secondary antibody was Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution. Rabbit monoclonal IgG (ab172730) (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control. 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, labeling Histone H3 (acetyl K27) with ab177178 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased nuclear staining in HeLa cells treated with TSA (500 ng/ml, 4 hours). The nuclear counter stain is DAPI (blue).

Immunohistochemical analysis of paraffin-embedded rat pancreas tissue labeling Histone H3 (acetyl K27) with ab177178 at 1/10000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was performed for 20 minutes. Nuclear staining on rat pancreas is observed. Counter stained with Hematoxylin.

The section was incubated with ab177178 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling Histone H3 (acetyl K27) with ab177178 at 1/10000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was performed for 20 minutes. Nuclear staining on mouse lung tissue is observed. Counter stained with Hematoxylin.

The section was incubated with ab177178 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human liver cancer tissue labeling Histone H3 (acetyl K27) with ab177178 at 1/1500 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was performed for 20 minutes. Nuclear staining on human liver cancer tissue is observed. Counter stained with Hematoxylin.

The section was incubated with ab177178 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

ab177178 was tested in Peptide array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
 
Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
 
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.