Anti-BNIP3 抗体 [EPR4034]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-BNIP3 antibody [EPR4034] (AB109362)

Immunocytochemistry/Immunofluorescence analysis of SH-SY5Y cells labelling BNIP3 with unpurified ab109362 at a 1/100 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BNIP3 antibody [EPR4034] (AB109362)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human renal adenocarcinoma tissue labelling BNIP3 with purified ab109362 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BNIP3 antibody [EPR4034] (AB109362)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling BNIP3 with unpurified ab109362 at a 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-BNIP3 antibody [EPR4034] (AB109362)

Lane 1 : Wild-type HAP1 whole cell lysate (40 μg)
Lane 2 : BNIP3 knockout HAP1 whole cell lysate (40 μg)
Lane 3 : SHSY5Y whole cell lysate (40 μg)
Lane 4 : A431 whole cell lysate (40 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109362 observed at 25 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab109362 was shown to recognize BNIP3 in wild-type HAP1 cells as signal was lost at the expected MW in BNIP3 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and BNIP3 knockout samples were subjected to SDS-PAGE. ab109362 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-BNIP3 antibody [EPR4034] (ab109362)

Predicted band size: 21 kDa

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• WB

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Western blot - Anti-BNIP3 antibody [EPR4034] (AB109362)

All lanes:

Western blot - Anti-BNIP3 antibody [EPR4034] (ab109362) at 1/1000 dilution

Lane 1:

Jurkat cell lysate treated with etoposide at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

SH-SY5Y cell lysate treated with camptothecin at 10 µg

Lane 4:

SH-SY5Y cell lysate at 10 µg

Lane 5:

MCF-7 cell lysate treated with Cocl2 at 10 µg

Predicted band size: 21 kDa

Observed band size: 30 kDa

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• WB

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Western blot - Anti-BNIP3 antibody [EPR4034] (AB109362)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-BNIP3 antibody [EPR4034] (ab109362) at 1/2000 dilution

Lane 1:

Rat kidney tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Mouse spleen tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 21 kDa

Observed band size: 30 kDa

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• WB

Supplier Data

Western blot - Anti-BNIP3 antibody [EPR4034] (AB109362)

The observed band sizes are consistent with publications. Bands around 30 and 60kDa should be monomer and dimer, respectively. Other bands should be different migrating forms (PMID : 15375294, PMID : 36396625, PMID : 9396766, PMID : 36396625, PMID : 33459136 etc.). 1% SDS hot lysis method could help to get stronger monomer forms. The protocol used for this test is available under the Support & downloads section

All lanes:

Western blot - Anti-BNIP3 antibody [EPR4034] (ab109362) at 1/1000 dilution

Lane 1:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate 20ug, prepared by RIPA method at 20 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate 20ug, prepared by 1% SDS hot method at 20 µg

Secondary

All lanes:

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

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Exposure time: 156s