All lanes : Anti-HMGB1 antibody [EPR21207] (ab213256) at 1 µg/ml

Lane 1 : NIH3T3 cell lysate
Lane 2 : MEF1 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : A431 cell lysate
Lane 5 : Human colon tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP conjugated Goat Anti-Human IgG (H+L) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 24 kDa


Exposure time: 20 minutes

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 40 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab213273 overnight at 4°C. Antibody binding was detected using a goat anti-human antibody conjugated to HRP, and visualised using ECL development solution ab133406.

IHC image of HMGB1 staining in a section of formalin-fixed paraffin-embedded normal mouse large intestine performed on a Leica BOND^TM. 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 ab213256, 1ug/ml, for 15 mins at room temperature.

An HRP-conjugated goat anti-Human IgG secondary was used 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 secondary-only 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.

 

IHC image of HMGB1 staining in a section of formalin-fixed paraffin-embedded normal human colon* performed on a Leica BOND^TM. 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 ab213256, 0.5ug/ml, for 15 mins at room temperature.

An HRP-conjugated goat anti-Human IgG secondary was used 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 secondary-only 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