Anti-NQO1 antibody [EPR3309]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-NQO1 antibody [EPR3309] (AB80588)

ab80588 staining NQO1in the human cell line MCF-7 (human breast carcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluorr^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NQO1 antibody [EPR3309] (AB80588)

Immunocytochemsitry/Immunofluorescence analysis of MCF-7 cells labelling NQO1 (green) with purified ab80588 at 1/80. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

• IP

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Immunoprecipitation - Anti-NQO1 antibody [EPR3309] (AB80588)

ab80588 (purified) at 1/50 immunoprecipitating NQO1 in HeLa cell lysate (Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-NQO1 antibody [EPR3309] (ab80588)

Predicted band size: 31 kDa

Observed band size: 31 kDa

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• WB

Lab

Western blot - Anti-NQO1 antibody [EPR3309] (AB80588)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-NQO1 antibody [EPR3309] (ab80588) at 1/25000 dilution

Lane 1:

SH-SY5Y cell lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 31 kDa

Observed band size: 31 kDa

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• WB

Lab

Western blot - Anti-NQO1 antibody [EPR3309] (AB80588)

Lanes 1 - 6 : Merged signal (red and green). Green - ab80588 observed at 31 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab80588 was shown to recognize NQO1 in wild-type HAP1 cells as signal was lost at the expected MW in NQO1 knockout cells. Wild-type and NQO1 knockout samples were subjected to SDS-PAGE. ab80588 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-NQO1 antibody [EPR3309] (ab80588) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 40 µg

Lane 2:

NQO1 knockout HAP1 whole cell lysate at 40 µg

Lane 3:

HepG2 whole cell lysate at 20 µg

Lane 4:

MEF1 whole cell lysate at 20 µg

Lane 5:

Human Kidney tissue lysate at 20 µg

Lane 6:

Mouse Kidney tissue lysate at 20 µg

Predicted band size: 31 kDa

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• WB

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Western blot - Anti-NQO1 antibody [EPR3309] (AB80588)

All lanes:

Western blot - Anti-NQO1 antibody [EPR3309] (ab80588) at 1/50000 dilution

Lane 1:

MCF7 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

A549 cell lysate at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 31 kDa

Observed band size: 31 kDa

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