Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) 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  ab8580 (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.

Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1 µg/ml + Calf thymus histone preparation (nuclear lysate) at 0.5 µg

Secondary
Goat Anti-Rabbit IgG (H+L) HRP- conjugated antibody at 1/50000 dilution

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 8 minutes

Human female lymphoblast immunostained with ab8580 (1:100) (yellowish green) specific for histone H3 lysine 4 (H3-K4) trimethylation; the DNA is stained red with propidium iodide (PI).

Note the inactive X chromosome (arrow) and pericentromeric heterochromatin are largely devoid of this modification.

IHC image of ab8580 staining Histone H3 (tri methyl K4) 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, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab8580, 1/500 dilution, 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 (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

ab8580 staining cultured human primary fibroblasts by ICC.

Cells were fixed in PFA and permeabilized in Triton X-100 and saponin prior to blocking with 1% BSA for 1 hour at RT.  The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 4°C.  An FITC-conjugated rabbit anti-rabbit IgG antibody was used as the secondary.

See Abreview

ab8580 staining Histone H3 (tri methyl K4) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

All 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 1/1000 and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with goat anti-rabbit Alexa-Fluor^®488 secondary (ab150077) at 2 μg/ml (shown in green) and goat anti-mouse Alexa-Fluor^®594 secondary (ab150120) at 2 μg/ml (shown in pseudo color 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 non-specific reaction between primary and secondary antibodies used.

All batches of ab8580 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 - tri methyl K4 peptide (ab1342), indicating that this antibody specifically recognises the Histone H3 - tri methyl K4 modification. Slight cross reactivity is observed with the Histone H3 - di methyl K4 modification. Optimization is recommended to avoid array signal saturation.

1. ab1340 - Histone H3 - mono methyl K4
2. ab1342 - Histone H3 - tri methyl K4
3. ab1771 - Histone H3 - mono methyl K9
4. ab1772 - Histone H3 - di methyl K9
5. ab1773 - Histone H3 - tri methyl K9
6. ab1780 - Histone H3 - mono methyl K27
7. ab1781 - Histone H3 - di methyl K27
8. ab1782 - Histone H3 - tri methyl K27
9. ab7228 - Histone H3 - unmodified
10. ab7768 - Histone H3 - di methyl K4

See Abreview

Staining (green) with the anti-trimethyl Lysine K4 of Histone H3 antibody (ab8580) shows ringing of regions that appear as nucleoplasmic holes. These represent the positions of splicing factor compartments, which are preferentially associated with active genes and highly acetylated histone H3.   

The antibody, as expected, fails to stain heterochromatin (red).