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RabMAb

Anti-Bak 抗体 [Y164] (ab32371)

製品の概要

  • 製品名
    Anti-Bak antibody [Y164]
    Bak 一次抗体 製品一覧
  • 製品の詳細
    Rabbit monoclonal [Y164] to Bak
  • 由来種
    Rabbit
  • 特異性
    ab32371 recognises Bak. The antibody does not cross-react with other Bcl2 members.
  • アプリケーション
    適用あり: WB, IHC-P, Flow Cyt, ICC/IF, IPmore details
  • 種交差性
    交差種: Human
  • 免疫原

    Synthetic peptide corresponding to Human Bak aa 1-100.

  • ポジティブ・コントロール
    • Hela cell lysate, human stomach carcinoma
  • 特記事項

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information. 

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

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. Avoid freeze / thaw cycle.
  • バッファー
    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.21% BSA
  • Concentration information loading...
  • 精製度
    Protein A purified
  • ポリ/モノ
    モノクローナル
  • クローン名
    Y164
  • アイソタイプ
    IgG
  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab32371 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
WB 1/10000. Predicted molecular weight: 23 kDa.

For unpurified use at 1/1000 - 1/5000. 

IHC-P 1/200.
Flow Cyt 1/10 - 1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/100.

For unpurified use at 1/250-1/500.

IP 1/20.

For unpurified use at 1/100. 

ターゲット情報

  • 機能
    In the presence of an appropriate stimulus, accelerates programmed cell death by binding to, and antagonizing the anti-apoptotic action of BCL2 or its adenovirus homolog E1B 19k protein. Low micromolar levels of zinc ions inhibit the promotion of apoptosis.
  • 組織特異性
    Expressed in a wide variety of tissues, with highest levels in the heart and skeletal muscle.
  • 配列類似性
    Belongs to the Bcl-2 family.
  • ドメイン
    Intact BH3 motif is required by BIK, BID, BAK, BAD and BAX for their pro-apoptotic activity and for their interaction with anti-apoptotic members of the Bcl-2 family.
  • 細胞内局在
    Mitochondrion membrane.
  • Information by UniProt
  • 参照データベース
  • 別名
    • Apoptosis regulator BAK antibody
    • BAK antibody
    • BAK like antibody
    • Bak NT antibody
    • BAK_HUMAN antibody
    • Bak1 antibody
    • Bcl 2 homologous antagonist/killer antibody
    • Bcl 2 like 7 protein antibody
    • Bcl-2 homologous antagonist/killer antibody
    • Bcl-2-like protein 7 antibody
    • BCL2 antagonist/killer 1 antibody
    • Bcl2 like 7 Protein antibody
    • Bcl2-L-7 antibody
    • BCL2L7 antibody
    • CDN1 antibody
    • Cell death inhibitor 1 antibody
    • MGC117255 antibody
    • MGC3887 antibody
    • NBak antibody
    • Pro apoptotic protein BAK antibody
    see all

画像

  • ab32371 (purified) at 1:20 dilution (2μg) immunoprecipitating Bak in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

    Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg
    Lane 2 (+): ab32371 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32371 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

    For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1:1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Bak with purified ab32371 at 1:20 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Bak with Purified ab32371 at 1:100 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200. ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human pancreatic carcinoma tissue sections labeling Bak with Purified ab32371 at 1:200 dilution (2.98 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
    secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human stomach tissue sections labeling Bak with Purified ab32371 at 1:200 dilution (2.98 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
    secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

  • All lanes : Anti-Bak antibody [Y164] (ab32371) at 1/10000 dilution (purified)

    Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
    Lane 2 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
    Lane 3 : Human fetal heart lysates

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

    Predicted band size: 23 kDa
    Observed band size: 23 kDa



    Blocking and diluting buffer: 5% NFDM/TBST

  • Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
    Lane 2: BAK knockout HAP1 whole cell lysate (20 µg)
    Lane 3: Human Heart whole cell lysate (20 µg)

    Lanes 1 - 3: Merged signal (red and green). Green - ab32371 observed at 25 kDa. Red - loading control, ab9484, observed at 37 kDa.

    Unpurified ab32371 was shown to specifically recognize BAK in wild-type HAP1 cells. No band was observed when BAK knockout cells were examined. Wild-type and BAK knockout samples were subjected to SDS-PAGE. Unpurified ab32371 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

  • Anti-Bak antibody [Y164] (ab32371) at 1/5000 dilution (unpurified) + HeLa cell lysate

    Predicted band size: 23 kDa
    Observed band size: 23 kDa

  • Unpurified ab32371 staining Bak in the human cell line HeLa (human cervix adenocarcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

    Isoytype control: Rabbit monoclonal IgG (Black)

    Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

  • Immunohistochemical analysis of Bak expression in paraffin embedded human stomach carcinoma, using 1/250 unpurified ab32371.

  • ICC/IF image of unpurified ab32371 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified ab32371, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Bak was immunoprecipitated from HCT116 p53-/- cell line whole cell lysate with unpurified ab32371 at 1/100 dilution.

    Western blot was performed from the immunoprecipitate using ab32371 at 1/2000 dilution.

    See Abreview

参考文献

This product has been referenced in:
  • Ruvolo PP  et al. Role of MSC-derived galectin 3 in the AML microenvironment. Biochim Biophys Acta 1865:959-969 (2018). Read more (PubMed: 29655803) »
  • Liu C  et al. MiR-144-3p promotes the tumor growth and metastasis of papillary thyroid carcinoma by targeting paired box gene 8. Cancer Cell Int 18:54 (2018). WB ; Human . Read more (PubMed: 29632436) »

See all 23 Publications for this product

レビューと Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (COLO205 colon carcinoma xenograft)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH 9
Permeabilization
No
Specification
COLO205 colon carcinoma xenograft
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 20°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

投稿 Dec 14 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample
Chinese Hamster Cell (CHO cell)
Specification
CHO cell
Permeabilization
Yes - 1% TRITON-X-100
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

投稿 Mar 18 2015

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

Abcam user community

Verified customer

投稿 Jul 30 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample
Rat Cell (h9c2)
Specification
h9c2
Permeabilization
Yes - 1% TRITON-X-100
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

投稿 Jul 30 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Sample
Mouse Cell (MEF)
Specification
MEF
Permeabilization
Yes - 1% TRITON-X-100
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

投稿 Jul 30 2014

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing
Sample
Rat Cell lysate - whole cell (h9c2)
Specification
h9c2
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

投稿 Jul 30 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Immuno-precipitation step
Protein A/G
Sample
Human Cell lysate - whole cell (HCT116 p53-/- cell line)
Specification
HCT116 p53-/- cell line
Total protein in input
100 µg
Username

Mr. Christian Marx

Verified customer

投稿 Aug 27 2013

The concentration of 1542-1 lot YI063010CS is 0.1160 mg/ml.

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - other (isolated mitochondria from NB4 cell line)
Loading amount
30 µg
Specification
isolated mitochondria from NB4 cell line
Gel Running Conditions
Reduced Denaturing (13%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Mr. Christian Marx

Verified customer

投稿 Feb 15 2013

Thank you for contacting us.  The purpose of the blocking step is to prevent non-specific binding between the antibodies and the tissue section, membrane, 96-well plate, etc.  It is possible to get a good signal without a blocking step, but blocking wi...

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

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