IHC image of histone H4 (acetyl K8) antibody staining in a section of formalin-fixed paraffin-embedded human breast adenocarcinoma* performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab15823, 0.5ug/ml, for 15 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.

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

Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 2 µg of ab15823 (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.

All lanes : Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (ab15823) at 1 µg/ml

Lane 1 : Sodium butyrate treated hela histone
Lane 2 : Untreated hela histone
Lane 3 : Sodium butyrate treated hela histone with Human Histone H4 (acetyl K8) peptide (ab15824) at 1 µg/ml
Lane 4 : Untreated hela histone with Human Histone H4 (acetyl K8) peptide (ab15824) at 1 µg/ml
Lane 5 : Sodium butyrate treated hela histone with Human Histone H4 peptide (ab15825) at 1 µg/ml
Lane 6 : Untreated hela histone with Human Histone H4 peptide (ab15825) at 1 µg/ml
Lane 7 : Sodium butyrate treated hela histone with Histone H4 peptide - acetyl K5 (ab15596) at 1 µg/ml
Lane 8 : Untreated hela histone with Histone H4 peptide - acetyl K5 (ab15596) at 1 µg/ml
Lane 9 : Sodium butyrate treated hela histone with Human Histone H4 (acetyl K12) peptide (ab15662) at 1 µg/ml
Lane 10 : Untreated hela histone with Human Histone H4 (acetyl K12) peptide (ab15662) at 1 µg/ml
Lane 11 : Sodium butyrate treated hela histone with Histone H2A peptide - acetyl K9 at 1 µg/ml
Lane 12 : Untreated hela histone with Histone H2A peptide - acetyl K9 at 1 µg/ml

Lysates/proteins at 2.5 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 11.2 kDa
Observed band size: 12 kDa

why is the actual band size different from the predicted?
Additional bands at: 22 kDa (possible cross reactivity)

The blot shows that a H4 peptide containing an acetylated K8 residue was able to block recognition of histones by ab15823. Peptides containing other acetylated K residues of H4 and H2A did not block binding of histones by ab15823. This suggests ab15823 specifically recognises Histone H4 acetyl K8.

ICC/IF image of ab15823 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab15823, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

All lanes : Anti-Histone H4 (acetyl K8) antibody - ChIP Grade (ab15823) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 peptide (ab15825) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 (acetyl K8) peptide (ab15824) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Histone H4 peptide - acetyl K5 at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 (acetyl K12) peptide (ab15662) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H2A (acetyl K9) peptide (ab54016) at 0.5 µg/ml

Lysates/proteins at 0.5 µg/ml per lane.

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

Performed under reducing conditions.

Predicted band size: 11.2 kDa
Observed band size: 13 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes