Anti-Nrf2 antibody (ab89443) at 1 µg/ml + Rat Testis at 50 µg

Secondary
Goat anti-mouse IgG-HRP at 1/5000 dilution

Predicted band size: 68 kDa

12.5% SDS-PAGE GEL

Anti-Nrf2 antibody (ab89443) at 1 µg/ml + PC12 cell lysate at 50 µg

Predicted band size: 68 kDa
Observed band size: 68 kDa

This image was generated using the ascites version of the product.

ICC/IF image of ab89443 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab89443, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

This image was generated using the ascites version of the product.

IHC image of Nrf2 staining in Human Lung formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. 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 ab89443, 0.5 µg/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.

This image was generated using the ascites version of the product.

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.

Overlay histogram showing HepG2 cells stained with ab89443 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab89443, 0.5µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
This image was generated using the ascites version of the product.

All lanes : Anti-Nrf2 antibody (ab89443) at 1 µg/ml

Lane 1 : Nrf2 transfected 293T cell lysate
Lane 2 : Non-transfected lysate

Lysates/proteins at 25 µg per lane.

Predicted band size: 68 kDa
Observed band size: 68 kDa
Additional bands at: 45 kDa. We are unsure as to the identity of these extra bands.

This image was generated using the ascites version of the product.

Anti-Nrf2 antibody (ab89443) at 1 µg/ml + recombinant immunogen at 0.2 µg

Predicted band size: 68 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?

Western blot against tagged recombinant immunogen using ab89443 antibody at 1µg/ml dilution. Predicted band size of immunogen is 11kDa.

This image was generated using the ascites version of the product.