製品の概要

  • 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
  • 特異性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 aln (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, as previously described. ... The expression of BNIP3 mRNA and protein was significantly reduced in the lungs of HET (as compared with WT) and Cre+ (as compared with Cre-) mice, demonstrating that BNIP3 expression is regulated by HIF-1 under physiological conditions in vivo."
  • アプリケーション適用あり: ICC, ELISA, IP, WB, IHC-P, Flow Cyt, ICC/IFmore details
  • 種交差性
    交差種: Mouse, Rat, Human
  • 免疫原

    Recombinant fragment (Residues 1-163 of 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

    BNIP3, formerly NIP3 (nineteen kDa interacting protein-3), is a pro-apoptotic, mitochondrial protein classified in the Bcl 2 family based on limited sequence homology to the Bcl 2 homology 3 (BH3) domain (amino acids 110-118) and C-terminal TM domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl 2, Bcl XL, CED9 and the adenovirus E1B 19K protein. Typically the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl 2/Bcl XL heterodimerization and confers pro-apoptotic activity. BNIP3 represents a subfamily of Bcl 2 related proteins, which functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl 2/Bcl XL interactions. The N-terminus (residues 1-49) and the C-terminus TM domain of BNIP3 are critical for Bcl 2 heterodimerization, and either region is sufficient for Bcl XL interaction. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. 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. Thus, the posttranslational control of BNIP3 expression through rapid protein degradation may constitute a mechanism for regulating the intracellular levels of a potentially lethal protein. Homologues of BNIP3 sharing both structural and functional similarity have been identified in mammals: Nix (also called BNIP3L/BNIP3alpha\/B5) and, in C. elegans, ceBNIP3. The TM domain of BNIP3 and Nix share 80% identity. Endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N-terminus in the cytoplasm and the C-terminus in the membrane during induction of cell death. This is accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial permeability transition (PT) pore, proton electrochemical gradient suppression, and increased reactive oxygen species production. BNIP3 has been reported to localize to the nuclear envelope when co-expressed with E1B 19K. Endogenous BNIP3 protein is abundant in murine and human skeletal muscle and is not detectable in lysates of all other nonskeletal muscle-bearing tissues and many cell lines, including myoblasts and differentiated myocytes.

     

    Alternative versions available:

    Anti-BNIP3 antibody (Alexa Fluor® 647) [ANa40] (ab196706)

    Anti-BNIP3 antibody (HRP) [ANa40] (ab196707)

製品の特性

  • 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Preservative: 15mM Sodium Azide
    Constituents: 0.01M PBS, pH 7.4
  • Concentration information loading...
  • 精製度Protein G purified
  • 一次抗体 備考BNIP3, formerly NIP3 (nineteen kDa interacting protein-3), is a pro-apoptotic, mitochondrial protein classified in the Bcl 2 family based on limited sequence homology to the Bcl 2 homology 3 (BH3) domain (amino acids 110-118) and C-terminal TM domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl 2, Bcl XL, CED9 and the adenovirus E1B 19K protein. Typically the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl 2/Bcl XL heterodimerization and confers pro-apoptotic activity. BNIP3 represents a subfamily of Bcl 2 related proteins, which functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl 2/Bcl XL interactions. The N-terminus (residues 1-49) and the C-terminus TM domain of BNIP3 are critical for Bcl 2 heterodimerization, and either region is sufficient for Bcl XL interaction. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. 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. Thus, the posttranslational control of BNIP3 expression through rapid protein degradation may constitute a mechanism for regulating the intracellular levels of a potentially lethal protein. Homologues of BNIP3 sharing both structural and functional similarity have been identified in mammals: Nix (also called BNIP3L/BNIP3alpha\/B5) and, in C. elegans, ceBNIP3. The TM domain of BNIP3 and Nix share 80% identity. Endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N-terminus in the cytoplasm and the C-terminus in the membrane during induction of cell death. This is accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial permeability transition (PT) pore, proton electrochemical gradient suppression, and increased reactive oxygen species production. BNIP3 has been reported to localize to the nuclear envelope when co-expressed with E1B 19K. Endogenous BNIP3 protein is abundant in murine and human skeletal muscle and is not detectable in lysates of all other nonskeletal muscle-bearing tissues and many cell lines, including myoblasts and differentiated myocytes.
  • 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

アプリケーション

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.

アプリケーション Abreviews 特記事項
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: 21.5 kDa).
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.

ターゲット情報

  • 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.
  • Information by UniProt
  • 参照データベース
  • 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

Anti-BNIP3 antibody [ANa40] 画像

  • Anti-BNIP3 antibody [ANa40] (ab10433) at 5 µg/ml + Human skeletal muscle tissue lysate - total protein (ab29330) at 20 µg

    Secondary
    Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 21.5 kDa
    Observed band size : 30 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 135 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 30 seconds
  • 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.
  • 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.

Anti-BNIP3 antibody [ANa40] (ab10433) 使用論文

This product has been referenced in:

See all 24 Publications for this product

Product Wall

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
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投稿 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
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投稿 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
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投稿 Jun 26 2015

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...

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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.

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

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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...

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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 ...

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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...

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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
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Dr. Sharon Sneddon

Verified customer

投稿 Nov 30 2011

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"