All lanes : Anti-Histone H3 (phospho T45) antibody (ab26127) at 1 µg

Lane 1 : HeLa treated with Calyculin A for 45min (0nM, untreated)
Lane 2 : HeLa treated with Calyculin A for 45min (20nM)
Lane 3 : HeLa treated with Calyculin A for 45min (20nM) with Human Histone H3 (phospho T45) peptide (ab26307) at 1 µg

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

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: 8 minutes

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 abX overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

ELISA using ab26127.

The red line indicates binding to the Histone H3 - Phospho T45 peptide (ab26307).

Binding to the following peptides was not observed:
Histone H3 (ab26308), Histone H3 peptide - phospho T3 (ab17578), Histone H3 peptide - phospho T11 (ab24444) or Histone H3 peptide - phospho T32 (ab14799)   

IHC image of ab26127 staining Histone H3 in Human normal 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 (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab26127, 7µg/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. No primary antibody was used in the negative control (shown on the 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