Anti-Nrf2 antibody [EP1808Y] - C-terminal (ab62352)

Blocking and dilution buffer: 5% NFDM/TBST.

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

Blocking and dilution buffer: 5% NFDM/TBST

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.

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.  

See Abreview

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.

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.

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).

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 ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1000).

Blocking and dilution buffer: 5% NFDM/TBST.

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.

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.