重组Anti-Bad抗体[Y208] (ab32445)
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, Human
概述
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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. -
Tested Applications & Species
Application Species Flow Cyt HumanICC/IF MouseIHC-P HumanWB Human -
免疫原
Synthetic peptide within Human Bad (N terminal). The exact sequence is proprietary.
(Peptide available asab206866) -
阳性对照
- WB: HAP1, MCF7 and HeLa cell lysate. IHC-P: Human lymphoma. ICC/IF: RAW 264.7. Flow Cyt: MCF7.
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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
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
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: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
Y208 -
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 lysates
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KO cell pellets
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Recombinant Protein
应用
The Abpromise guarantee
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.
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
|
ICC/IF |
Mouse
|
IHC-P |
Human
|
WB |
Human
|
All applications |
Rat
Dog
|
应用 | Ab评论 | 说明 |
---|---|---|
WB | (7) |
1/2000 - 1/5000. Detects a band of approximately 23 kDa (predicted molecular weight: 18 kDa).
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IHC-P | (1) |
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) |
1/250 - 1/500.
|
Flow Cyt | (1) |
1/20 - 1/50.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
说明 |
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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
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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
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数据库链接
- Entrez Gene: 483763 Dog
- Entrez Gene: 572 Human
- Entrez Gene: 12015 Mouse
- Entrez Gene: 64639 Rat
- Omim: 603167 Human
- SwissProt: Q92934 Human
- SwissProt: Q61337 Mouse
- SwissProt: O35147 Rat
see all -
别名
- AI325008 antibody
- BAD antibody
- BAD_HUMAN antibody
see all
图片
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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
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- 2: Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab32445 was shown to react with Bad in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264843 (knockout cell lysate ab256847) was used. Wild-type HeLa and BAD 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. ab32445 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 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-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.
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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 kDaLanes 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.
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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.
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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? -
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.
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.
数据表及文件
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SDS download
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Datasheet download
文献 (73)
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