All lanes : HRP Anti-Histone H3 (phospho S10 + T11) antibody [E173] (ab200616) at 1/5000 dilution

Lane 1 : HeLa Histone Preparation Nuclear Lysate
Lane 2 : HeLa Histone Preparation Nuclear Lysate - Colcemid Treated

Lysates/proteins at 2.5 µg per lane.

Developed using the ECL technique.

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: 1 second

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab200616 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

IHC image of Histone H3 (phospho S10) staining in a section of formalin-fixed paraffin-embedded human colon adenocarcinoma*, performed on a Leica BOND. 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 ab200616, 1/100 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

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