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Validated using a knockout cell line

Anti-BNIP3 antibody [ANa40] (ab10433)

Reviews (8)Q&A (6)References (103)
Western blot - Anti-BNIP3 antibody [ANa40] (ab10433)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BNIP3 antibody [ANa40] (ab10433)
  • Western blot - Anti-BNIP3 antibody [ANa40] (ab10433)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BNIP3 antibody [ANa40] (ab10433)
  • Flow Cytometry - Anti-BNIP3 antibody [ANa40] (ab10433)

Key features and details

  • Mouse monoclonal [ANa40] to BNIP3
  • Suitable for: WB, IHC-P, Flow Cyt
  • Knockout validated
  • Reacts with: Human
  • Isotype: IgG2b

Conjugates logo Related conjugates and formulations

Alexa Fluor® 647 HRP

Overview

  • Product name

    Anti-BNIP3 antibody [ANa40]
    See all BNIP3 primary antibodies

  • Description

    Mouse monoclonal [ANa40] to BNIP3

  • Host species

    Mouse

  • Tested applications

    Suitable for: WB, IHC-P, Flow Cytmore details

  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat

  • Immunogen

    Recombinant fragment corresponding to Human BNIP3.

  • Epitope

    The epitope recognized by the antibody resides within amino acids 112-124 of human BNIP3 molecule.

  • Positive control

    • This antibody gave a positive signal in human skeletal muscle tissue lysate in western blot and on human kidney formalin-fixed, paraffin-embedded tissue sections in IHC.

  • General notes

    This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab10433 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB (5)
Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 30 kDa (predicted molecular weight: 30 kDa).

We recommend using 3% milk as the blocking agent for Western blot.
IHC-P
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt
Use 0.1-1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
Notes
WB
Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 30 kDa (predicted molecular weight: 30 kDa).

We recommend using 3% milk as the blocking agent for Western blot.
IHC-P
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt
Use 0.1-1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

Target

  • Function

    Apoptosis-inducing protein that, which can overcome BCL2 suppression. May play a role in repartitioning calcium between the two major intracellular calcium stores in association with BCL2.

  • Sequence similarities

    Belongs to the NIP3 family.

  • Cellular localization

    Mitochondrion. Mitochondrion membrane. Coexpression with the EIB 19-kDa protein results in a shift in NIP3 localization pattern to the nuclear envelope. Colocalizes with ACAA2 in the mitochondria.
  • Information by UniProt

  • Database links

    • Alternative names

      • BCL2 Adenovirus E1B 19kDa Interacting Protein 3 antibody
      • BCL2/adenovirus E1B 19 kDa protein interacting protein 3 antibody
      • BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 antibody
      • BNIP 3 antibody
      • BNIP3 antibody
      • BNIP3_HUMAN antibody
      • NIP 3 antibody
      • NIP3 antibody
      see all

    Images

    • Western blot - Anti-BNIP3 antibody [ANa40] (ab10433)
      Western blot - Anti-BNIP3 antibody [ANa40] (ab10433)

      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)

      Lanes 1 - 3: Merged signal (red and green). Green - ab10433 observed at 30 kDa. Red - loading control, ab181602, observed at 37 kDa.

      ab10433 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. ab10433 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 5 µg/mL and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BNIP3 antibody [ANa40] (ab10433)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BNIP3 antibody [ANa40] (ab10433)Image from Jin T et al., J Transl Med. 2012 Mar 30;10:64 Fig 1.; doi:10.1186/1479-5876-10-64; 30 March 2012, Journal of Translational Medicine 2012, 10:64
      Immunohistochemical analysis of Human laryngeal squamous cell carcinoma tissue, staining BNIP3 with ab10433.

      Antigen retrieval was performed by heat mediation in citrate buffer. The sections were incubated with primary antibody (1/100) overnight at 4°C in a humidified chamber. Staining was visualized using DAB, followed by hematoxylin nuclear counterstaining.
    • Western blot - Anti-BNIP3 antibody [ANa40] (ab10433)
      Western blot - Anti-BNIP3 antibody [ANa40] (ab10433)
      Anti-BNIP3 antibody [ANa40] (ab10433) at 1 µg/ml + Human skeletal muscle tissue lysate - total protein (ab29330) at 20 µg

      Secondary
      Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/5000 dilution

      Performed under reducing conditions.

      Predicted band size: 30 kDa


      Exposure time: 5 seconds


      This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab10433 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406. 

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BNIP3 antibody [ANa40] (ab10433)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BNIP3 antibody [ANa40] (ab10433)

      IHC image of BNIP3 antibody staining in a section of formalin-fixed paraffin-embedded normal human kidney* performed on a Leica BONDTM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab10433, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

      *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

    • Flow Cytometry - Anti-BNIP3 antibody [ANa40] (ab10433)
      Flow Cytometry - Anti-BNIP3 antibody [ANa40] (ab10433)

      Overlay histogram showing HepG2 cells stained with ab10433 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab10433, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

    References (103)

    Publishing research using ab10433? Please let us know so that we can cite the reference in this datasheet.

    ab10433 has been referenced in 103 publications.

    • Vara-Pérez M  et al. BNIP3 promotes HIF-1a-driven melanoma growth by curbing intracellular iron homeostasis. EMBO J 40:e106214 (2021). PubMed: 33932034
    • Huo S  et al. Alleviation of Inflammation and Oxidative Stress in Pressure Overload-Induced Cardiac Remodeling and Heart Failure via IL-6/STAT3 Inhibition by Raloxifene. Oxid Med Cell Longev 2021:6699054 (2021). PubMed: 33824698
    • Zhu HL  et al. Melatonin protects against environmental stress-induced fetal growth restriction via suppressing ROS-mediated GCN2/ATF4/BNIP3-dependent mitophagy in placental trophoblasts. Redox Biol 40:101854 (2021). PubMed: 33454563
    • Wang M  et al. S-Nitroso-L-Cysteine Ameliorated Pulmonary Hypertension in the MCT-Induced Rats through Anti-ROS and Anti-Inflammatory Pathways. Oxid Med Cell Longev 2021:6621232 (2021). PubMed: 33574976
    • Xu S  et al. EBV-LMP1 promotes radioresistance by inducing protective autophagy through BNIP3 in nasopharyngeal carcinoma. Cell Death Dis 12:344 (2021). PubMed: 33795637
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-10 of 14 Abreviews or Q&A

    Western blot abreview for Anti-BNIP3 antibody [ANa40]

     Average
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Dopaminergic Neurons)
    Gel Running Conditions
    Reduced Denaturing (NuPage 4-12% MES buffer)
    Loading amount
    30 µg
    Specification
    Dopaminergic Neurons
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Oct 22 2020

    Western blot abreview for Anti-BNIP3 antibody [ANa40]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (colon cancer)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    30 µg
    Specification
    colon cancer
    Blocking step
    Milk as blocking agent for 3 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Oct 13 2020

    Western blot abreview for Anti-BNIP3 antibody [ANa40]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Cancer cell lines)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    30 µg
    Specification
    Cancer cell lines
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jan 15 2020

    Western blot abreview for Anti-BNIP3 antibody [ANa40]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (MCF-7)
    Gel Running Conditions
    Reduced Denaturing (10)
    Loading amount
    35 µg
    Treatment
    20nM siNeg or siBNIP3 for 48 and 72h
    Specification
    MCF-7
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jan 14 2019

    Immunocytochemistry/ Immunofluorescence abreview for Anti-BNIP3 antibody [ANa40]

     Excellent
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Monkey Cell (Kidney)
    Permeabilization
    Yes - 0.1% triton x-100
    Specification
    Kidney
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jun 08 2016

    Immunohistochemistry (Frozen sections) abreview for Anti-BNIP3 antibody [ANa40]

     Average
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Brain)
    Permeabilization
    No
    Specification
    Brain
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Mar 04 2016

    Question

    lease find enclosed the detail concerning ab10433 and the western blots.



    1) Abcam product code ab10433



    2) Abcam order reference number or product batch number 1090379 lot

    3) Description of the problem
    Many bands are observed on westernblot. It looks like a coomassie staining rather than a westernblot. This antibody (1/1000) was tested on total lysate of HeLa, MEFs (WT and SV40), HCT116, U2OS, Raji, Ramos, Jurkat, Namalwa. I tried monoQc and RipA lysis buffer and the result is the same. Blocking was done in milk 5% and the secondary antibody used is Goat anti-mouse Alexa Fluor680. Wash steps were performed in TBS tween 0.1% for at least 1h.





    4) Sample preparation:

    Type of sample (whole cell lysates, fraction, recombinant protein…): Whole lysate from cell lines mentioned above.

    Lysis buffer : MonoQc and RIPA

    Protease inhibitors: Roche protease inhibitor EDTA free

    Phosphatase inhibitors none

    Reducing agent β-mercaptoethanol

    Boiling for ≥5 min? yes

    Protein loaded ug/lane or cells/lane 30ug per lane

    Positive control 0

    Negative control 0



    5) Percentage of gel 10%

    Type of membrane Nitrocellulose

    Protein transfer verified Ponceau staining

    Blocking agent and concentration non-fat milk 5% in TBS 0.1% Tween

    Blocking time 1h

    Blocking temperature RT



    6) Primary antibody (If more than one was used, describe in “additional notes”) :

    Concentration or dilution 1/1000

    Diluent buffer non-fat milk 5% in TBS 0.1% tween

    Incubation time overnight

    Incubation temperature: 4°C



    7) Secondary antibody:

    Species: goat

    Reacts against: mouse

    Concentration or dilution 1/5000

    Diluent buffer non-fat milk 5% in TBS 0.1% tween

    Incubation time 1h

    Incubation temperature: RT

    Fluorochrome or enzyme conjugate: Fluorochrome for Odyssey



    8) Washing after primary and secondary antibodies:

    Buffer TBS 0.1% tween

    Number of washes 3



    9)Detection method Licor odyssey

    10) How many times have you run this staining? 3 times for this primary antibody

    Do you obtain the same results every time? Yes, there is no specific band but a lot of unspecific

    What steps have you altered to try and optimize the use of this antibody? I changed the lysis buffer

    Read More
    Answer

    Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

    The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

    I would like to reassure you that ab10433 is tested and covered by our 6 month guarantee for use inWB and human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

    Reviewing this case, I would like to offer some suggestions to help optimise the results from ab10433.

    1.) I suggest to purify mitochondria instead of using crude extract to enrich the protein. It seems to be weakly expressed.

    2.) BNIP3 contains PEST sequences suggesting that the protein may be susceptible to rapid degradation by proteases. PEST sequences commonly contain high local concentrations of amino acids P, E, S, T, and D flanked by charged amino acids and these are abundantly present in NIP3.
    I therefore recommend to block the proteasom with a inhibitor, for example mg132, to avoid BNIP3 degradation.

    3.) It seems that BNIP3 has to be induced. maybe this publication will provide more information:

    Bellot G et al. Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol 29:2570-81 (2009).

    4.) I also suggest to use a positive control. We know of customers that have good results with PC3 cells.

    5.) In my opinion the background bands just come up since the protein is not there and the blot is slightly over exposed. Maybe a no primary control will clarify if the secondary is responsible for that.

    I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

    Read More

    Question

    Thankyou for getting back to me. . On this particular order, we ordered Bcl2 and BNIP3; the Bcl2 antibody works fine. However, the BNIP3 antibody has yet to give us a signal. It is definitley not of the quality of previous BNIP3 antibody that we have received from your company. Please send the replacement to the following address.

    Thankyou in advance

    Read More
    Answer

    I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1126945.

    This replacement vial will be shipped Monday to arrive Tuesday, July 17. To check the status of the order please contact our Customer Service team and reference this number.

    Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

    I wish you the best of luck with your research.

    Read More

    Question

    I have now run several gels with protein lysates that very recently gave us very strong BNIP3 signal. I have utilized your antibody at 1:500 with little to no signal at the 50-60kd BNIP3 dimer and absolutely no 30 kd signal. I am sure that this antibody is not of the past quality of antibodies I have received from your company and would very much appreciate a replacement and preferablly a different lot. thankyou and I look forward to hearing from you.

    Read More
    Answer

    I agree this vial of ab10433 you have received is not working as expected. I would be happy to send a free replacement.

    So I can arrange the shipment, please provide the PO number or Abcam order reference number associated with the purchase of this product.

    Thanks in advance for the additional information!

    Read More

    Question

    WB with MCF7 cells (hypoxic and normoxic conditions)
    200 ug protein loaded
    Ab: 1/1000 overnight 4 degree
    Problem: no signal

    suggested to try 1/500 as per datasheet recommendations

    Read More
    Answer

    According to our records, ab10433 was proving difficult to use in WB and we were in contact in order to help resolve the issue.

    Looking at our correspondence, it appears that we are awaiting more details to resolve this case.

    As we discussed over the phone, please let me know if a 1/500 dilution helped to improve the results. If not, I'd be happy to offer either a free of charge replacement, a credit or refund.


    If the requested information has already been sent, it appears that it did not reach our Scientific Support team and we apologize for this inconvenience. In this case we would like to ask for the information again so that we can reach a resolution.

    If the issue has already been settled, please let us know so that we can be assured that the problem has been solved to your satisfaction and update our records.

    We wish you the best of luck with your research and look forward to a reply.

    Read More

    1-10 of 14 Abreviews or Q&A

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