使用敲除细胞株进行验证重组RabMAb

重组Anti-Bad抗体[Y208] (ab32445)

Reviews (13)Q&A (4)References (73)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [Y208] to Bad
  • Suitable for: WB, IHC-P, ICC/IF, Flow Cyt
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

概述

  • 产品名称

    Anti-Bad抗体[Y208]
    参阅全部 Bad 一抗

  • 描述

    兔单克隆抗体[Y208] to Bad

  • 宿主

    Rabbit

  • 特异性

    The antibody does not cross-react with other Bcl-2 members.

  • 经测试应用

    适用于: WB, IHC-P, ICC/IF, Flow Cytmore details

  • 种属反应性

    与反应: Mouse, Rat, Human
    预测可用于: Dog

  • 免疫原

    Synthetic peptide within Human Bad (N terminal). The exact sequence is proprietary.
    (Peptide available as ab206866)

  • 阳性对照

    • WB: HAP1, MCF7 and HeLa cell lysate. IHC-P: Human lymphoma. ICC/IF: RAW 264.7. Flow Cyt: MCF7.

  • 常规说明

    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
    For more information see here.

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

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    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, as well as customer reviews and Q&As.

性能

应用

Our Abpromise guarantee covers the use of ab32445 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
WB 1/2000 - 1/5000. Detects a band of approximately 23 kDa (predicted molecular weight: 18 kDa).
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/250 - 1/500.
Flow Cyt 1/20 - 1/50.

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

靶标

  • 功能

    Promotes cell death. Successfully competes for the binding to Bcl-X(L), Bcl-2 and Bcl-W, thereby affecting the level of heterodimerization of these proteins with BAX. Can reverse the death repressor activity of Bcl-X(L), but not that of Bcl-2 (By similarity). Appears to act as a link between growth factor receptor signaling and the apoptotic pathways.

  • 组织特异性

    Expressed in a wide variety of tissues.

  • 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.

  • 翻译后修饰

    Phosphorylated on one or more of Ser-75, Ser-99, Ser-118 and Ser-134 in response to survival stimuli, which blocks its pro-apoptotic activity. Phosphorylation on Ser-99 or Ser-75 promotes heterodimerization with 14-3-3 proteins. This interaction then facilitates the phosphorylation at Ser-118, a site within the BH3 motif, leading to the release of Bcl-X(L) and the promotion of cell survival. Ser-99 is the major site of AKT/PKB phosphorylation, Ser-118 the major site of protein kinase A (CAPK) phosphorylation. Ser-75 is phosphorylated by AKT/PKB, protein kinase A and PIM2.

  • 细胞定位

    Mitochondrion outer membrane. Cytoplasm. Upon phosphorylation, locates to the cytoplasm.
  • Information by UniProt

  • 数据库链接

    see all

  • 别名

    • AI325008 antibody
    • BAD antibody
    • BAD_HUMAN antibody
    • BBC 2 antibody
    • BBC2 antibody
    • BBC6 antibody
    • Bcl 2 Antagonist of Cell Death antibody
    • Bcl 2 Binding Component 6 antibody
    • BCL X / BCL 2 Binding Protein antibody
    • BCL X Binding Protein antibody
    • Bcl XL/Bcl 2 Associated Death Promoter antibody
    • Bcl-2-binding component 6 antibody
    • Bcl-2-like protein 8 antibody
    • Bcl-XL/Bcl-2-associated death promoter antibody
    • Bcl2 antagonist of cell death antibody
    • BCL2 antagonist of cell death protein antibody
    • BCL2 associated agonist of cell death antibody
    • Bcl2 Associated Death Promoter antibody
    • BCL2 binding component 6 antibody
    • BCL2 binding protein antibody
    • Bcl2 Like 8 Protein antibody
    • Bcl2-L-8 antibody
    • BCL2L8 antibody
    • Proapoptotic BH3 Only Protein antibody
    see all

图片

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bad antibody [Y208] (ab32445)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bad antibody [Y208] (ab32445)

    Immunohistochemical staining of paraffin-embedded human lymphoma using ab32445 at 1/100 dilution.

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Western blot - Anti-Bad antibody [Y208] (ab32445)
    Western blot - Anti-Bad antibody [Y208] (ab32445)
    All lanes : Anti-Bad antibody [Y208] (ab32445) at 1/2000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : BAD knockout HeLa cell lysate
    Lane 3 : Wild-type HAP1 cell lysate
    Lane 4 : BAD knockout HAP1 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 18 kDa
    Observed band size: 23 kDa
    why is the actual band size different from the predicted?



    Lanes 1-4: Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - loading control ab8245 observed at 37 kDa.

     ab32445 Anti-Bad antibody [Y208] was shown to specifically react with BAD in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264843 (knockout cell lysate ab256847) was used. Wild-type and BAD knockout samples were subjected to SDS-PAGE. ab32445 and Anti-GAPDH antibody [6C5] - Loading Control were incubated overnight at 4°C at 1 in 2000 dilution and 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.

     

  • Western blot - Anti-Bad antibody [Y208] (ab32445)
    Western blot - Anti-Bad antibody [Y208] (ab32445)
    All lanes : Anti-Bad antibody [Y208] (ab32445) at 1/2000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : BAD knockout HAP1 whole cell lysate
    Lane 3 : HeLa whole cell lysate
    Lane 4 : MCF7 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 18 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab32445 was shown to specifically recognise BAD in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when BAD knockout cells were examined. Wild-type and BAD knockout samples were subjected to SDS-PAGE. Ab32445 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 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.

  • Immunocytochemistry/ Immunofluorescence - Anti-Bad antibody [Y208] (ab32445)
    Immunocytochemistry/ Immunofluorescence - Anti-Bad antibody [Y208] (ab32445)

    Immunocytochemical analysis of 2% paraformaldehyde-fixed,0.1% Triton-X100 permeabilized RAW 264.7 labeling Bad with ab32445 at 1/250dilution,followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 568) secondary antibody at 1/1000 dilution.

  • Western blot - Anti-Bad antibody [Y208] (ab32445)
    Western blot - Anti-Bad antibody [Y208] (ab32445)
    Anti-Bad antibody [Y208] (ab32445) at 1/5000 dilution + Hela cell lysate

    Predicted band size: 18 kDa
    Observed band size: 23 kDa why is the actual band size different from the predicted?

  • Flow Cytometry - Anti-Bad antibody [Y208] (ab32445)
    Flow Cytometry - Anti-Bad antibody [Y208] (ab32445)
    Overlay histogram showing MCF-7 cells stained with ab32445 (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 (ab32445, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in MCF-7 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • Anti-Bad antibody [Y208] (ab32445)
    Anti-Bad antibody [Y208] (ab32445)

实验方案

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

文献 (73)

发表研究结果有使用 ab32445？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab32445 被引用在 73 文献中.

  • Li Q  et al. Silencing of synaptotagmin 13 inhibits tumor growth through suppressing proliferation and promoting apoptosis of colorectal cancer cells. Int J Mol Med 45:234-244 (2020). PubMed: 31939613
  • Zheng J  et al. MSCs ameliorate hepatocellular apoptosis mediated by PINK1-dependent mitophagy in liver ischemia/reperfusion injury through AMPKa activation. Cell Death Dis 11:256 (2020). PubMed: 32312955
  • Hu J  et al. ERO1a inhibits cell apoptosis and regulates steroidogenesis in mouse granulosa cells. Mol Cell Endocrinol 511:110842 (2020). PubMed: 32376276
  • Liu K  et al. Protective effects of calbindin-D28K on the UVB radiation-induced apoptosis of human lens epithelial cells. Int J Mol Med 45:1793-1802 (2020). PubMed: 32236567
  • Feng C  et al. Neuroprotective Effect of Danhong Injection on Cerebral Ischemia-Reperfusion Injury in Rats by Activation of the PI3K-Akt Pathway. Front Pharmacol 11:298 (2020). PubMed: 32218735
View all Publications for this product

客户评价及客户问答

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1-10 of 17 Abreviews or Q&A

Western blot abreview for Anti-Bad antibody [Y208]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (SKOV-3)
Gel Running Conditions
Reduced Denaturing (10)
Loading amount
20 µg
Specification
SKOV-3
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Read More

Abcam user community

Verified customer

提交于 Aug 19 2019

Western blot abreview for Anti-Bad antibody [Y208]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Human Tissue lysate - whole (Heart)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Specification
Heart
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Read More

Abcam user community

Verified customer

提交于 Dec 24 2018

Western blot abreview for Anti-Bad antibody [Y208]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Rat Tissue lysate - whole (Heart)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Specification
Heart
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Read More

Abcam user community

Verified customer

提交于 Dec 24 2018

Immunocytochemistry abreview for Anti-Bad antibody [Y208]

 Good
Abreviews
abreview image
Application
Immunocytochemistry
Sample
Rat Cultured Cells (Cardiomyocytes)
Permeabilization
Yes - Triton x-100, 0.03%
Specification
Cardiomyocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

提交于 Feb 28 2018

Question

Please send the IgG concentrations of the following lots.
ab33906 lot YH090207C -
ab33862 lot YH082410D -
ab32429 lot YG080503 -
ab40660 lot YI070802CS -
ab32445 lot YI072804DS -

Read More
Answer

ab33906 lot YH090207C - 0.3260 mg/ml

ab33862 lot YH082410D - 0.6380 mg/ml

ab32429 lot YG080503 - 0.0790 mg/ml

ab40660 lot YI070802CS - 0.1030 mg/ml

ab32445 lot YI072804DS - 0.0250 mg/ml

Read More

Immunocytochemistry abreview for Anti-Bad antibody [Y208]

 Good
Abreviews
abreview image
Application
Immunocytochemistry
Sample
Mouse Cultured Cells (RAW 264.7)
Specification
RAW 264.7
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton-X100 in 2% BSA for 15min
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
Read More

Dr. Mahesh Shivananjappa

Verified customer

提交于 May 19 2012

Question

Dear Technical service,
Which antibodies could you recommended as a 2nd step to the following products:
ab32445 and ab33862 for flow cytometry?
Awaiting your answer.
Best regards.

Read More
Answer

Thank you for contacting us.
There is a wide range of secondaries suitable to detect either ab32445 or ab33862 in Flow Cytometry. I have conducted a search through our Secondaries search tool in the Abcam Website and found more than 100 matches, depending on the conjugation dye.
I would recommend you to have a look at the results and depending on the wavelength you are interested in measuring, choose the option that better matches your requirements.
https://www.abcam.com/index.html?pageconfig=searchresults&search=IgG&pt=7&sk=app&sv=99&sn=Flow Cyt&l=3&fViewMore=1
If you have any doubt about any of the products identified in the search, you need a proforma, or require further assistance, please do not hesitate to contact me back.

Read More

Question

Ordered ab90435 by mistake and would like to return it.

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Answer

Thank you for contacting Abcam.

Included in this email are the instructions for returning your item to Abcam. When you return the goods, please ensure that you follow the procedures as below:

- Return in the original Abcam box with the cooler box and ice pack (or similar) within 7 days from today.
- Return all documents (or copies of) that arrived with the product (if they’re available – should be invoice, delivery note and datasheet)
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- Ship to the following address:

Attn: Returns ORDER REF:
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Please be advised that a 20% restock fee will apply to this order. The most up-to-date information regarding your order, invoice, and credit note status is available online in your Abcam account information. If your order was placed by credit card, your refund will be issued to your credit card. If your order was placed by Purchase Order, all customers must submit a credit note with their invoice to their Accounts Payable department to be handled. The Accounts Payable must have a copy of the credit note so the original invoice can be updated to reflect the correct amount owed.

If you have any other questions please do not hesitate to contact us!

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Customer Service hours of operation:
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Western blot abreview for Anti-Bad antibody [Y208]

 Good
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Glioblastoma neurospheres cells (primary))
Loading amount
30 µg
Specification
Glioblastoma neurospheres cells (primary)
Gel Running Conditions
Reduced Denaturing (12.5% gel)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
Read More

Abcam user community

Verified customer

提交于 Dec 06 2011

Western blot abreview for Anti-Bad antibody [Y208]

 Average
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Platelets)
Loading amount
20 µg
Specification
Platelets
Treatment
Calcium Ionophore for 1 h
Gel Running Conditions
Reduced Denaturing (4-20%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Read More

Dr. Mahesh Shivananjappa

Verified customer

提交于 Nov 14 2011

1-10 of 17 Abreviews or Q&A

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