Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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

ab62352 staining Nrf2 in untreated wild type A549 cells (left panel), treated wild type A549 cells (middle panel) and untreated NFE2L2 knockout A549 cells (right panel). Cells were treated with 2µM of MG-132 for 18 hours (ab141003). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab62352 at 0.2 µg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2µg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2µg/ml (shown in magenta). Nuclear DNA was labelled in blue with DAPI.

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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).

• WB

Lab

Western blot - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

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

False colour image of Western blot : Anti-Nrf2 antibody [EP1808Y] - ChIP Grade staining at 1/1000 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.

Target band (indicated by arrow) was observed at 85-90 kDa in wild-type A549 cell lysates with no signal observed at this size in NFE2L2 knockout cell line ab285359 (knockout cell lysate ab289682). A band lower than the target was present on both WT and KO. According to the literature (PMID : 40043449), the lower band is calmegin.

To generate this image, wild-type and NFE2L2 knockout A549 cell lysates were analysed. Please note that MG132 treatment does not affect expression levels of Nrf2. 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Nrf2 antibody [EP1808Y] (<a href='/en-us/products/primary-antibodies/nrf2-antibody-ep1808y-ab62352'>ab62352</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 Vehicle control MG132 (0 uM, 18 h) cell lysate at 20 µg

Lane 2:

Wild-type A549 Treated MG132 (2 uM, 18 h) cell lysate at 20 µg

Lane 3:

NFE2L2 [21] knockout A549 Vehicle control MG132 (0 uM, 18 h) cell lysate at 20 µg

Lane 4:

NFE2L2 [21] knockout A549 Treated MG132 (2 uM, 18 h) cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 68 kDa

false

• WB

Lab

Western blot - Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (AB180845)

All lanes:

Western blot - Anti-Nrf2 antibody [EP1808Y] (<a href='/en-us/products/primary-antibodies/nrf2-antibody-ep1808y-ab62352'>ab62352</a>) at 1/500 dilution

Lane 1:

Western blot - Human NFE2L2 (Nrf2) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-nfe2l2-nrf2-knockout-hela-cell-lysate-ab263934'>ab263934</a>)

Lane 1:

Wild-type HeLa control MG132 (0 uM, 18 h) cell lysate

Lane 2:

Wild-type HeLa treated MG132 (2 uM, 18 h) cell lysate

Lane 2:

Western blot - Human NFE2L2 (Nrf2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-nfe2l2-nrf2-knockout-hela-cell-line-ab262507'>ab262507</a>)

Lane 3:

NFE2L2 knockout HeLa control MG132 (0 uM, 18 h) cell lysate

Lane 4:

NFE2L2 knockout HeLa treated MG132 (2 uM, 18 h) cell lysate

Lane 5:

HCT 116 cell lysate

false