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.

ab4729 staining Histone H3 (acetyl K27) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were incubated with 10 mM sodium butyrate (ab120948) for 6 hours (Treated) or solvent-only for control purposes (Non-treated). Cells were fixed with 100% methanol (5 minutes) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1 hour. The cells were then incubated with ab4729 at 0.5 µg/ml and ab7291 at 1 µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a anti-rabbit AlexaFluor^®488 secondary antibody (ab150077) at 2 μg/ml (shown in green) and a goat anti-mouse AlexaFluor^®594 (ab150120) at 2 μg/ml (shown in pseudo colour red). Nuclear DNA was labeled in blue with DAPI.

Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

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

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

All lanes : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 1/2500 dilution

Lane 1 : Untreated Mouse MEF cell lysate
Lane 2 : 0.4 µM Trichostatin A treatment for 18 hr Mouse MEF cell lysate

Lysates/proteins at 9 µg per lane.

Secondary
All lanes : Donkey Anti-Rabbit IgG H&L (HRP) (ab6802) at 1/20000 dilution

Predicted band size: 15 kDa

See Abreview

Primary antibody: ab4729 (H3 acetyl K27)
Dilution: 1/100

ab4729 strongly stained histones of mouse ES cells. However, fluroescence was greatly diminished following pre-blocking using a H3 acetyl K9 peptide. This suggests the antibody cross-reacts with the K9 and K27 residues.

 

Lanes 1 & 3 : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 0.2 µg/ml
Lanes 2 & 4 : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 0.1 µg/ml

Lanes 1-2 : Calf thymus histone lysate
Lanes 3-4 : Calf thymus histone lysate with Human Histone H3 (acetyl K27) peptide (ab24404) at 2 µg

Lysates/proteins at 1 µg per lane.

Secondary
All lanes : Goat anti-rabbit (HRP) at 1/2000 dilution

Predicted band size: 15 kDa
Observed band size: 17 kDa

why is the actual band size different from the predicted?



ab4729 specifically recognises acetyl K27 histone H3 in catlf thymus histone lysate, which is specifically blocked using the immunizing peptide ab24404.

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.

Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 1 µg/ml + HeLa (Human epithelial cell line from cervix adenocarcinoma) histone preparation, nuclear Lysate - Butyrate treated at 2.5 µg

Secondary
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
Additional bands at: 17 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 10 seconds