Western blot - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

All lanes:

Western blot - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1 µg/m

All lanes:

Calf thymus histone preparation (nuclear lysate) at 0.5 µg

Secondary

All lanes:

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

Exposure time: 8min

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

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

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

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.