Overview

  • Product name
    Anti-BNIP3 antibody [ANa40]
    See all BNIP3 primary antibodies
  • Description
    Mouse monoclonal [ANa40] to BNIP3
  • Host species
    Mouse
  • Specificity
    This antibody was originally tested on whole cell lysate from 293T cells transfected with BNIP3. Levels of constitutively expressed BNIP3 are very variable and a good positive control is strongly recommended. Zhang et al. (2008) comment "Very low levels of BNIP3 protein were detected by immunoblot assay of lysates prepared from WT MEFs that were cultured at 20% O2, whereas hypoxia strongly induced expression of BNIP3 protein, which migrated as a 30-kDa monomer and 60-kDa dimer...BNIP3 expression is regulated by HIF-1 under physiological conditions in vivo."
  • Tested applications
    Suitable for: ICC, ELISA, IP, WB, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment (Residues 1-163 of 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

    Western blot protocol advice:
    We recommend using 3% milk as the blocking agent for Western blot.

    This antibody clone is manufactured by Abcam. 

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

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
ICC Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
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.

ICC/IF Use at an assay dependent concentration.

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

  • 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.

  • 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.
  • 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

  • IHC image of ab10433 staining in human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab10433, 5µg/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.

  • ab10433 staining BNIP3 in Human fibroblasts by Immunocytochemistry/ Immunofluorescence. Cells were fixed in 2% paraformaldehyde for 30 minutes at room temperature and cells were treated with 50 mM NH4Cl in PBS for 10 minutes to quench free aldehyde groups after permeabilisation. Cells were then permeabilized in 0.1% Triton X-100 prior to blocking in 1% BSA for 1 hour at room temperature. The primary antibody was diluted 1/400 and incubated with the sample for 1 hour at room temperature. The secondary antibody was Alexa Fluor® 555-conjugated goat anti-mouse polyclonal, diluted 1/1000.

    See Abreview

  • 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

This product has been referenced in:
  • Yuan C  et al. BNIP3/Bcl-2-mediated apoptosis induced by cyclic tensile stretch in human cartilage endplate-derived stem cells. Exp Ther Med 15:235-241 (2018). Read more (PubMed: 29375685) »
  • Sato Y  et al. Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle. FASEB J 32:1428-1439 (2018). Read more (PubMed: 29127187) »
See all 68 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Jun 08 2016

Abcam has not validated the combination of species/application used in this Abreview.
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

Abcam user community

Verified customer

Submitted Mar 04 2016

Application
Western blot
Sample
Chinese Hamster Cell lysate - whole cell (CHO cell)
Gel Running Conditions
Reduced Denaturing
Loading amount
30 µg
Specification
CHO cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jun 26 2015

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

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

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

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

Answer

Thank you for contacting us.

I am sorry to hear that the antibody is not working as before.

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.

Ilook forward to hear back from you.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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 1087951.

To check the status of the order please contact our Customer Service team and reference this number.

(Please note that the lot that is giving you the multiple bands, GR4979, is a higher concentration of IgG (2.4 mg/ml) than the lot that gave you a cleaner result. However, if you are not seeing a relatively intense band at the expected size, then diluting GR4979 further, for instance 1/2500, will not improve the result).

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
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Fibroblasts)
Specification
Fibroblasts
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C

Dr. Sharon Sneddon

Verified customer

Submitted Nov 30 2011

1-10 of 11 Abreviews or Q&A

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