Name: Anti-BNIP3 antibody [ANa40]
SKU: ab10433
Rating: 4 (8 reviews)

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Western blot - 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

Alexa Fluor® 647 HRP

製品名

Anti-BNIP3 antibody [ANa40]
BNIP3 一次抗体製品一覧
製品の詳細

Mouse monoclonal [ANa40] to BNIP3
由来種

Mouse
アプリケーション

適用あり: WB, IHC-P, Flow Cytmore details
種交差性

交差種: Human
交差が予測される動物種: Mouse, Rat
免疫原

Recombinant fragment corresponding to Human BNIP3.
エピトープ

The epitope recognized by the antibody resides within amino acids 112-124 of human BNIP3 molecule.
ポジティブ･コントロール
- 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.
特記事項

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

製品の状態

Liquid
保存方法

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
バッファー

pH: 7.40
Preservative: 0.02% Sodium azide
Constituents: PBS, 6.97% L-Arginine
Concentration information loading...
精製度

Protein G purified
ポリ/モノ

モノクローナル
クローン名

ANa40
ミエローマ

unknown
アイソタイプ

IgG2b
軽鎖の種類

kappa
研究分野

Alternative Versions
- Alexa Fluor® 647 Anti-BNIP3 antibody [ANa40] (ab196706)
- HRP Anti-BNIP3 antibody [ANa40] (ab196707)
Compatible Secondaries
Isotype control
- Mouse IgG2b, kappa monoclonal [7E10G10] - Isotype Control (ab170192)
Recombinant Protein
- Recombinant Mouse BNIP3 protein (His tag) (ab235015)

The Abpromise guarantee

Abpromise保証は、次のテスト済みアプリケーションにおけるab10433の使用に適用されます

アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。

アプリケーション	Abreviews	特記事項
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.

特記事項
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.

機能

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.
配列類似性

Belongs to the NIP3 family.
細胞内局在

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.
Target information above from: UniProt accession Q12983 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
参照データベース
- Entrez Gene: 664 Human
- Entrez Gene: 12176 Mouse
- Entrez Gene: 84480 Rat
- Omim: 603293 Human
- SwissProt: Q12983 Human
- SwissProt: O55003 Mouse
- Unigene: 144873 Human
- Unigene: 378890 Mouse
別名
- 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

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)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)

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)

IHC image of BNIP3 antibody staining in a section of formalin-fixed paraffin-embedded normal human kidney* performed on a Leica BOND^TM 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)

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.

Mouse on Mouse staining protocol

Click here to view the general protocols

SDS download

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Datasheet download

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ab10433 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab10433 は 103 報の論文で使用されています。

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

レビューと Q&A

Show All レビュー Q&A

レビューを送る質問を送る

1-10 of 14 Abreviews or Q&A

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

Abcam user community

Verified customer

投稿 Oct 22 2020

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

Abcam user community

Verified customer

投稿 Oct 13 2020

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

Abcam user community

Verified customer

投稿 Jan 15 2020

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

Abcam user community

Verified customer

投稿 Jan 14 2019

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

投稿 Jun 08 2016

Application

Immunohistochemistry (Frozen sections)

Sample

Mouse Tissue sections (Brain)

Permeabilization

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

投稿 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

Abcam community

Verified customer

Asked on Jul 24 2012

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.

Abcam Scientific Support

Answered on Jul 24 2012

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

Abcam community

Verified customer

Asked on Jul 13 2012

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.

Abcam Scientific Support

Answered on Jul 13 2012

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.

Abcam community

Verified customer

Asked on Jul 10 2012

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!

Abcam Scientific Support

Answered on Jul 10 2012

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

Abcam community

Verified customer

Asked on Jul 07 2012

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.

Abcam Scientific Support

Answered on Jul 07 2012

1-10 of 14 Abreviews or Q&A

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Key features and details

Related conjugates and formulations

製品の概要

製品名

製品の詳細

由来種

アプリケーション

種交差性

免疫原

エピトープ

ポジティブ･コントロール

特記事項

製品の特性

製品の状態

保存方法

バッファー

精製度

ポリ/モノ

クローン名

ミエローマ

アイソタイプ

軽鎖の種類

研究分野

関連製品

Alternative Versions

Compatible Secondaries

Isotype control

Recombinant Protein

アプリケーション

The Abpromise guarantee

ターゲット情報

機能

配列類似性

細胞内局在

参照データベース

別名

画像

プロトコール

データシートおよび資料

SDS download

Datasheet download

参考文献 (103)

レビューと Q&A

Western blot abreview for Anti-BNIP3 antibody [ANa40]

Western blot abreview for Anti-BNIP3 antibody [ANa40]

Western blot abreview for Anti-BNIP3 antibody [ANa40]

Western blot abreview for Anti-BNIP3 antibody [ANa40]

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

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