Recombinant Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (ab180845)

This data was developed using ab62352, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concnetration: 5% NFDM/TBST.

ab181602 was used as GAPDH loading control.

The two bands are different isoforms of Nrf2, the molecular weight observed is consistent with what has been described in the literature: PMID: 17512459, PMID: 22703241.

False colour image of Western blot: Anti-Nrf2 antibody [EP1808Y] - ChIP Grade staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab62352 was shown to bind specifically to Nrf2. A band was observed at 85 kDa in wild-type HeLa cell lysates with no signal observed at this size in NFE2L2 CRISPR-Cas9 edited cell line ab262507 (CRISPR-Cas9 edited cell lysate ab263934). The band observed in the CRISPR-Cas9 edited lysate lane below 85 kDa is likely to represent a truncated form of Nrf2. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and NFE2L2 CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreatic carcinoma tissue labelling Nrf2 with purified ab62352 at a dilution of 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab62352).

Clone EP1808Y (ab180845) has been successfully conjugated by Abcam. This image was generated using Anti-Nrf2 antibody [EP1808Y] (Alexa Fluor® 647). Please refer to ab194985 for protocol details.

ab194985 staining Nrf2 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab194985 at a working dilution 1/100 (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 2µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product gave a positive signal in 4% formaldehyde (10 min) fixed HeLa cells under the same testing conditions.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using ab62352, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure:
Lanes 1 and 2: 15 seconds.
Lanes 3 and 4: 3 minutes.

Clone EP1808Y (ab180845) has been successfully conjugated by Abcam. This image was generated using Anti-Nrf2 antibody [EP1808Y] (PE). Please refer to ab223926 for protocol details. 

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab223926 (red line). The cells were fixed with 4% formaldehyde (10 minutes) and then permeabilized with 0.1% PBS-Triton X-100 for 15 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab223926, 1/5000 dilution) for 30 minutes at 22°C. 

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabeled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter. 

This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 minutes)/permeabilized with 0.1% PBS-Triton X-100 for 15 minutes used under the same conditions. 

Chromatin was prepared from HepG2 cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab62352 (blue), and 20µl of A/G sepharose bead slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab62352).

Clone EP1808Y (ab180845) has been successfully conjugated by Abcam. This image was generated using Anti-Nrf2 antibody [EP1808Y] (Alexa Fluor® 488). Please refer to ab194984 for protocol details.

ab194984 staining Nrf2 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab194984 at a working dilution of 1/100 (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 594, shown in red) at 2µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Nrf2 with purified ab62352 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. The cells were co-stained with ab195889, an Alexa Fluor^® 594-conjugated mouse anti-alpha tubulin antibody (1/200). Nuclei counterstained with DAPI (blue).

Secondary antibody only control: PBS was used instead of the primary antibody as the negative control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab62352).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney cancer tissue sections labeling Nrf2 with ab62352 at 1/100 dilution. The tissue was fixed with paraformaldehyde and a heat mediated antigen retrival step was performed with TRIS-EDTA Buffer pH 9.0. Staining with ab62352 at 1/100 was carried out in a dilution buffer with blocking for 30 minutes at 20°C. A undiluted goat anti-rabbit HRP conjugated secondary antibody was used.  

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab62352).

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Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Nrf2 with purified ab62352 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody (1/1000) was used as the secondary antibody. Cells were counterstained with ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594). DAPI was used to stain the nuclei blue.

Secondary antibody only control: PBS was used instead of the primary antibody as the negative control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab62352).

ab62352 (purified) at 1/30 immunoprecipitating Nrf2 in SAOS-2 whole cell lysate.

Lane 1 (input): SAOS-2 whole cell lysate (10µg)
Lane 2 (+): ab62352 + SAOS-2 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab62352 in SAOS-2 whole cell lysate.

For western blotting, ab62352 was used at a dilution of 1/500 and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking and dilution buffer: 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab62352).

Intracellular Flow Cytometry analysis of HeLa cells labelling  Nrf2 with purified ab62352 at a dilution of 1/60 (red). Cells were fixed with 4% paraformaldehyde. Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody  (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab62352). 

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling  Nrf2 with ab62352 at 1/40 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor^®488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab62352).