ChIP - Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab4729 (blue), and 20µl 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)

IHC image of Histone H3 (acetyl K27) staining in human breast carcinoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab4729, 1µg/ml, for 8 mins 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.

Western blot - Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)

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

Lane 1 : HeLa Histone Preparation Nuclear Lysate - Butyrated
Lane 2 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - unmodified R17 at 0.5 µg/ml
Lane 3 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K9 (ab16635) at 0.5 µg/ml
Lane 4 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K14 at 0.5 µg/ml
Lane 5 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K18 (ab24003) at 0.5 µg/ml
Lane 6 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K23 (ab48359) at 0.5 µg/ml
Lane 7 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K27 (ab24404) at 0.5 µg/ml
Lane 8 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H3 peptide - acetyl K36 at 0.5 µg/ml
Lane 9 : HeLa Histone Preparation Nuclear Lysate - Butyrated with Histone H4 peptide - acetyl K12 (ab15662) at 0.5 µg/ml

Lysates/proteins at 2.5 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 15.4 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)
Additional bands at : 12 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 150 seconds

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)

ab4729 (1/500) staining Histone H3 (acetyl K27) in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please see abreview.

Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

Immunofluorescence - Histone H3 (acetyl K27) antibody (ab4729)

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.

Petra Hajkova - Gurdon Institute, Cambridge University

Western blot - Histone H3 (acetyl K27) antibody (ab4729)

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

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

Lysates/proteins at 1 µg per lane.

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

Predicted band size : 15.4 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.

Western blot - Histone H3 (acetyl K27) antibody (ab4729)

All lanes : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729) at 0.2 µg/ml

Lane 1 : HeLa whole cell lysate
Lane 2 : HeLa nuclear lysate
Lane 3 : HeLa whole cell lysate with Histone H3 peptide (ab17163) at 1 µg/ml
Lane 4 : HeLa nuclear lysate with Histone H3 peptide (ab17163) at 1 µg/ml
Lane 5 : HeLa whole cell lysate with Histone H3 peptide - acetyl K27 (ab24404) at 1 µg/ml
Lane 6 : HeLa nuclear lysate with Histone H3 peptide - acetyl K27 (ab24404) at 1 µg/ml
Lane 7 : HeLa whole cell lysate with Histone H3 peptide - acetyl K9 (ab16635) at 1 µg/ml
Lane 8 : HeLa nuclear lysate with Histone H3 peptide - acetyl K9 (ab16635) at 1 µg/ml
Lane 9 : HeLa whole cell lysate with Histone H3 peptide - acetyl K18 (ab24003) at 1 µg/ml
Lane 10 : HeLa nuclear lysate with Histone H3 peptide - acetyl K18 (ab24003) at 1 µg/ml
Lane 11 : HeLa whole cell lysate with Histone H3 peptide - asymmetric di methyl R26 (ab2854) at 1 µg/ml
Lane 12 : HeLa nuclear lysate with Histone H3 peptide - asymmetric di methyl R26 (ab2854) at 1 µg/ml
Lane 13 : HeLa whole cell lysate with Histone H4 peptide - acetyl K12 (ab15662) at 1 µg/ml
Lane 14 : HeLa nuclear lysate with Histone H4 peptide - acetyl K12 (ab15662) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size : 15.4 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)


ab4729 recognises acetyl K27 histone H3 in HeLa whole cell extracts (lane1), which is efficiently blocked using the immunizing peptide ab24404 (lane5). ab4729 is not blocked using acetyl K9 histone H3 (lane7), acetyl K18 histone H3 (lane9), dimethyl R26 histone H3 (lane 11)or acetyl K12 histone H4 (lane 13) peptides.

However, ab4729 is partially blocked by the unmodified peptide (lane 3). This indicates that the ab4729 mainly recognises acetyl K27 histone H3 but also to a small degree recognises unmodified histone H3.

ab4729 does not appear to detect acetyl K27 histone H3 in HeLa nuclear extracts (lanes 2,4,6,8,10,12,14).

Immunocytochemistry/ Immunofluorescence - Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)

ICC/IF image of ab4729 stained Hela cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab4729, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.