Anti-Nrf2 antibody [EP1808Y]

• WB

Lab

Western blot - Anti-Nrf2 antibody [EP1808Y] (AB62352)

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] (ab62352) 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

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Nrf2 antibody [EP1808Y] (AB62352)

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] (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 Fluorr^®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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Nrf2 antibody [EP1808Y] (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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Nrf2 antibody [EP1808Y] (AB62352)

ab62352 staining Nrf2 in untreated HeLa cells (top panel) and treated HeLa cells (bottom 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 1μg/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 red). 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] (AB62352)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Nrf2 antibody [EP1808Y] (AB62352)

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.

• WB

Lab

Western blot - Anti-Nrf2 antibody [EP1808Y] (AB62352)

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 knockout cell line ab262507 (knockout cell lysate ab263934). The band observed in the knockout 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 knockout 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.

All lanes:

Western blot - Anti-Nrf2 antibody [EP1808Y] (ab62352) 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

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• WB

Lab

Western blot - Anti-Nrf2 antibody [EP1808Y] (AB62352)

The expression of Nrf2 is stimulated by oxidative stress, electrophiles and chemical activators (PMID : 25761198, PMID : 27638861 and PMID : 28587109). ab62352 detects no signal in most of the untreated samples in WB. Stimuli treated samples are recommended.

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

All lanes:

Western blot - Anti-Nrf2 antibody [EP1808Y] (ab62352) at 1/200 dilution

Lane 1:

HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

HCT 116 (Human colorectal carcinoma epithelial cell) treated with 25uM MG-132 for 4 hours whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 68 kDa

Observed band size: 100 kDa

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Exposure time: 60s

• WB

AbReview81245****

Western blot - Anti-Nrf2 antibody [EP1808Y] (AB62352)

Western blot analysis using ab62352 at 1 : 1000 on Human iPSC-cardiomyocytes. Blocking agent and dilution buffer was 5% Skim Milk in TBS-Tween.

A cellular fractionation in Human iPSC-cardiomyocyte cells was performed to separate the nucleus from the cytoplasm (Lane 2).

All lanes:

Western blot - Anti-Nrf2 antibody [EP1808Y] (ab62352) at 1/1000 dilution

Lane 1:

Human iPSC-cardiomyocytes cytoplasmatic fraction, at 20 µg

Lane 2:

Human iPSC-cardiomyocytes nuclear fraction at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/2000 dilution

Observed band size: 100 kDa

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