Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: NQO1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HepG2 whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab28947 observed at 31 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab28947 was shown to specifically react with NQO1 in wild-type HAP1 cells as signal was lost in NQO1 knockout cells. Wild-type and NQO1 knockout samples were subjected to SDS-PAGE. ab28947 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Lane 1 : Marker
Lane 2 : Anti-NQO1 antibody [A180] (ab28947) at 1 µg/ml

Lane 2 : Kidney (Human) Tissue Lysate (ab7920) at 20 µg

Secondary
Lane 2 : IRDye 680 Conjugated Goat Anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 30 kDa
Observed band size: 30 kDa

IHC image of NQO1 staining in sections of formalin fixed paraffin embedded normal human pancreas* (left) and human pancreas adenocarcinoma* (right), performed on a Leica BOND^TM 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 ab28947, 0.1µg/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.

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

ICC/IF image of ab28947 stained human HEK 293 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab28947, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

Overlay histogram showing HeLa cells stained with ab28947 (red line). The cells were fixed with 80% methanol (5 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 (ab28947, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was goat anti-mouse DyLight® 488 (IgG H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Sandwich ELISA for the detection of NQO1. ab28947 (1/500) was used as the capture antibody. A rabbit polyclonal raised againts the C-terminal end of NQO1 was used for the detection. Please refer to abreview for further experimental details.

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IHC image of NQO1 staining in human breast adenocarcinoma 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 ab28947, 0.1µg/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.

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.

Sandwich ELISA for the detection of NQO1, using ab28947 (1/500) as the capture antibody and ab34173 (1/1000) for the detection.

Human breast cancer tissue stained with ab28947 NQO1 antibody.

Immunohistochemical analysis of dog skin tissue, staining NQO1 with ab28947.

Tissue was fixed with formaldehyde and antigen retrieval was by heat mediation in a citrate buffer (pH 6). Samples were incubated with primary antibody (1/175 in BSA in TBS) for 45 minutes. ab98784 rabbit polyclonal to anti-mouse HRP (IgG (1/500) was used as the secondary antibody.

See Abreview