All lanes : Anti-HMGB1 antibody [EPR21210] (ab227526) at 1/1000 dilution

Lane 1 : Wild-type Hap1 whole cell lysate
Lane 2 : HMGB1 knockout Hap1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : Jurkat whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 24 kDa
Observed band size: 24 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab227526 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab227526 was shown to recognize HMGB1 in wild-type Hap1 cells as signal was lost at the expected MW in HMGB1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HMGB1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab227526 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-HMGB1 antibody [EPR21210] (ab227526) at 1 µg/ml

Lane 1 : NIH3T3 cell lysate
Lane 2 : MEF1 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 10 µg per lane.

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

Performed under reducing conditions.

Predicted band size: 24 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?
Additional bands at: 125 kDa (possible cross reactivity)


Exposure time: 30 seconds

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 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 ab227526 overnight at 4°C. Antibody binding was detected using a goat anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

IHC image of HMGB1 staining in human normal colon formalin fixed paraffin embedded tissue section, performed on a Leica BOND^TM system using the standard protocol B. 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 ab227526, 0.05µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. 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 ab227526, 0.05ug/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