重组Anti-Bak抗体[Y164] (ab32371)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y164] to Bak
- Suitable for: WB, IHC-P, Flow Cyt, ICC/IF
- Knockout validated
- Reacts with: Human
概述
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产品名称
Anti-Bak抗体[Y164]
参阅全部 Bak 一抗 -
描述
兔单克隆抗体[Y164] to Bak -
宿主
Rabbit -
特异性
ab32371 recognises Bak. The antibody does not cross-react with other Bcl2 members. -
Tested Applications & Species
Application Species Flow Cyt HumanICC/IF HumanIHC-P HumanIP HumanWB Human -
免疫原
Synthetic peptide corresponding to Human Bak aa 1-100.
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阳性对照
- WB: Recombinant Human Bak protein (ab114337), HAP1, HeLa and HEK-293 cell lysates; human heart and fetal heart lysates. IHC-P: Human pancreatic carcinoma, stomach carcinoma and normal stomach tissue. ICC/IF: HeLa cells. IP: HCT 116 and HeLa cell lysates. Flow cyt: HeLa cells.
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常规说明
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
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.
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 the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.
One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.
Learn more here.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.21% BSA -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
Y164 -
同种型
IgG -
研究领域
相关产品
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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KO cell pellets
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Positive Controls
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Recombinant Protein
应用
The Abpromise guarantee
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.
Tested applications are guaranteed to work and covered by our Abpromise guarantee.
Predicted to work for this combination of applications and species but not guaranteed.
Does not work for this combination of applications and species.
应用 | Species |
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Flow Cyt |
Human
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ICC/IF |
Human
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IHC-P |
Human
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IP |
Human
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WB |
Human
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应用 | Ab评论 | 说明 |
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WB | (1) |
1/10000. Predicted molecular weight: 23 kDa.
For unpurified use at 1/1000 - 1/5000. |
IHC-P | (1) |
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
|
Flow Cyt |
1/10 - 1/20.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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ICC/IF |
1/100.
For unpurified use at 1/250-1/500. |
说明 |
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WB
1/10000. Predicted molecular weight: 23 kDa. For unpurified use at 1/1000 - 1/5000. |
IHC-P
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
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. |
靶标
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功能
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. -
组织特异性
Expressed in a wide variety of tissues, with highest levels in the heart and skeletal muscle. -
序列相似性
Belongs to the Bcl-2 family. -
结构域
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. -
细胞定位
Mitochondrion membrane. - Information by UniProt
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数据库链接
- Entrez Gene: 578 Human
- Omim: 600516 Human
- SwissProt: Q16611 Human
- Unigene: 485139 Human
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别名
- Apoptosis regulator BAK antibody
- BAK antibody
- BAK like antibody
see all
图片
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All lanes : Anti-Bak antibody [Y164] (ab32371) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BAK1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 23 kDaLanes 1- 2: Merged signal (red and green). Green - ab32371 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab32371 was shown to react with Bak in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265277 (knockout cell lysate ab257077) was used. Wild-type HeLa and BAK1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32371 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bak antibody [Y164] (ab32371)
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. -
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.
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All lanes : Anti-Bak antibody [Y164] (ab32371)
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : BAK knockout HAP1 whole cell lysate
Lane 3 : Human Heart whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 23 kDaLanes 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.
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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 Detection Reagent (HRP) (ab131366) was used for detection 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).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bak antibody [Y164] (ab32371)
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 kDaBlocking and diluting buffer: 5% NFDM/TBST
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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)
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bak antibody [Y164] (ab32371)
Immunohistochemical analysis of Bak expression in paraffin embedded human stomach carcinoma, using 1/250 unpurified ab32371.
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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.
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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.
数据表及文件
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SDS download
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Datasheet download
文献 (40)
ab32371 被引用在 40 文献中.
- Xue YN et al. Zinc and p53 disrupt mitochondrial binding of HK2 by phosphorylating VDAC1. Exp Cell Res 374:249-258 (2019). PubMed: 30528266
- Zahid KR et al. Novel tumor suppressor SPRYD4 inhibits tumor progression in hepatocellular carcinoma by inducing apoptotic cell death. Cell Oncol (Dordr) 42:55-66 (2019). PubMed: 30238408
- Zhang B et al. The Ibr-7 derivative of ibrutinib exhibits enhanced cytotoxicity against non-small cell lung cancer cells via targeting of mTORC1/S6 signaling. Mol Oncol 13:946-958 (2019). PubMed: 30663221
- Zamorano-León JJ et al. Effect of Pectin on the Expression of Proteins Associated with Mitochondrial Biogenesis and Cell Senescence in HT29-Human Colorectal Adenocarcinoma Cells. Prev Nutr Food Sci 24:187-196 (2019). PubMed: 31328124
- Shi B et al. Upregulation of JHDM1D-AS1 protects PDLSCs from H2O2-induced apoptosis by decreasing DNAJC10 via phosphorylation of eIF2a. Biochimie 165:48-56 (2019). PubMed: 31276733