重组Anti-Smad2 (phospho T8) + Smad3 (phospho T8)抗体[EPR23682-64] (ab254407)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR23682-64] to Smad2 (phospho T8) + Smad3 (phospho T8)
- Suitable for: ICC/IF, Dot blot, IP, ChIC/CUT&RUN-seq, WB
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
概述
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产品名称
Anti-Smad2 (phospho T8) + Smad3 (phospho T8)抗体[EPR23682-64] -
描述
兔单克隆抗体[EPR23682-64] to Smad2 (phospho T8) + Smad3 (phospho T8) -
宿主
Rabbit -
经测试应用
适用于: ICC/IF, Dot blot, IP, ChIC/CUT&RUN-seq, WBmore details
不适用于: Flow Cyt or IHC-P -
种属反应性
与反应: Mouse, Rat, Human -
免疫原
This product was produced with the following immunogens:
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers. -
阳性对照
- WB: RAW264.7, PC-12, HaCaT, and HaCaT ( treated with 100nM calyculin A for 30 min) whole cell lysates; Human liver cancer tissue lysate; Mouse lung and liver tissue lysates. ICC/IF: HaCaT and RAW264.7 cells. IP: HaCaT whole cell lysate. ChIC/CUT&RUN-Seq: HaCaT cells.
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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.
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 long term. Avoid freeze / thaw cycle. -
存储溶液
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% 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
EPR23682-64 -
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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Related Products
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab254407于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。
应用 | Ab评论 | 说明 |
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ICC/IF |
1/50.
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Dot blot |
1/1000.
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IP |
1/30.
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ChIC/CUT&RUN-seq |
Use at an assay dependent concentration.
5µg |
|
WB |
1/1000. Detects a band of approximately 55, 60 kDa.
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说明 |
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ICC/IF
1/50. |
Dot blot
1/1000. |
IP
1/30. |
ChIC/CUT&RUN-seq
Use at an assay dependent concentration. 5µg |
WB
1/1000. Detects a band of approximately 55, 60 kDa. |
靶标
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细胞定位
Smad2: Cytoplasm. Nucleus. Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4. On dephosphorylation by phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1. Smad3: Cytoplasm. Nucleus. Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4 (PubMed:15799969). Through the action of the phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1 (PubMed:16751101, PubMed:19289081). Co-localizes with LEMD3 at the nucleus inner membrane (PubMed:15601644). MAPK-mediated phosphorylation appears to have no effect on nuclear import (PubMed:19218245). PDPK1 prevents its nuclear translocation in response to TGF-beta (PubMed:17327236). -
数据库链接
- Entrez Gene: 4087 Human
- Entrez Gene: 4088 Human
- Entrez Gene: 17126 Mouse
- Entrez Gene: 17127 Mouse
- Entrez Gene: 25631 Rat
- Entrez Gene: 29357 Rat
- Omim: 601366 Human
- Omim: 603109 Human
see all
图片
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ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HaCaT (human skin keratinocyte) cells and 5µg of ab254407 [EPR23682-64]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.
Additional screenshots of mapped reads can be downloaded here.
The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 cells labelling Smad2 (phospho T8) + Smad3 (phospho T8) with ab254407 at 1/50 (9.34 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing weak cytoplasmic and nuclear staining in RAW 264.7 cells, while strong cytoplasmic and weak nuclear staining in RAW 264.7 cells treated with calyculin A (100 nM) for 30 mins is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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All lanes : Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution
Lane 1 : HaCaT (human skin keratinocyte), whole cell lysate at 10 µg
Lane 2 : HaCaT - phosphatase treated membrane, whole cell lysate at 10 µg
Lanes 3 & 5 : HaCaT whole cell lysate at 20 µg
Lane 4 : HaCaT treated with 100nM calycin A for 30 min, whole cell lysate at 20 µg
Lane 6 : HaCaT treated with /ml TGF beta1 for 24h, whole cell lysate at 20 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Observed band size: 55,60 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The molecular weight observed is consistent with what has been described in the literature. (PMID: 25998442, 30666129).
Calyculin A is a known phosphatase inhibitor, which increased the level of pSmad2/3 (T8). The shifted band after treated with Calyculin A might be due to multiple phosphorylation events.
The down-regulation of pSmad2 (T8) is induced by TGF-beta1 treatment in HaCaT (PMID: 19201832).
Bands between 15-25kDa may be caused by degradation.
Exposure time: Lane 1, 2: 26 secondsLane 3, 4: 59 secondsLane 5, 6: 3 minutes
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Smad2 (phospho T8) + Smad3 (phospho T8) was immunoprecipitated from 0.35 mg HaCaT (human skin keratinocyte), whole cell lysate with ab254407 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254407 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: HaCaT (human skin keratinocyte), whole cell lysate 10 ug
Lane 2: ab254407 IP in HaCaT whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254407 in HaCaT whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 110 seconds
The molecular weight observed is consistent with what has been described in the literature. (PMID: 25998442, 30666129).
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Dot blot analysis of Smad2 (phospho T8) + Smad3 (phospho T8) using ab254407 at 1/1000 (0.467 ug/ml) dilution followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.
Exposure time: 3 minutes.
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Lane 1: Smad2/3 peptide (aa 6-14)
Lane 2: Smad2/3 (phospho T8) peptide (aa 2-10 )
Lane 3: Smad2/3 peptide (aa 2-14)
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HaCaT cells labelling Smad2 (phospho T8) + Smad3 (phospho T8) with ab254407 at 1/50 (9.34 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing weak cytoplasmic and nuclear staining in HaCaT cells, while strong cytoplasmic and weak nuclear staining in HaCaT cells treated with calyculin A (100 nM) for 30 mins is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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All lanes : Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution
Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 2 : RAW264.7 - phosphatase treated membrane, whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution
Observed band size: 60 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Exposure time: 26 seconds.
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All lanes : Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution
Lane 1 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 2 : PC-12 - phosphatase treated membrane, whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution
Observed band size: 60 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The molecular weight observed is consistent with what has been described in the literature. (PMID: 25998442, 30666129)
Exposure time: 59 seconds.
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All lanes : Anti-Smad2 (phospho T8) + Smad3 (phospho T8) antibody [EPR23682-64] (ab254407) at 1/1000 dilution
Lane 1 : Human liver cancer tissue lysate
Lane 2 : Mouse lung tissue lysate
Lane 3 : Mouse liver tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution
Observed band size: 55,60 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The molecular weight observed is consistent with what has been described in the literature. (PMID: 25998442, 30666129).
Bands between 15-25kDa may be caused by degradation.
Exposure time: 3 minutes.
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
Certificate of Compliance
文献 (6)
ab254407 被引用在 6 文献中.
- Li Q et al. Naringenin inhibits autophagy and epithelial-mesenchymal transition of human lens epithelial cells by regulating the Smad2/3 pathway. Drug Dev Res 83:389-396 (2022). PubMed: 34402084
- Wu Y et al. LOXL2 Inhibitor Attenuates Angiotensin II-Induced Atrial Fibrosis and Vulnerability to Atrial Fibrillation through Inhibition of Transforming Growth Factor Beta-1 Smad2/3 Pathway. Cerebrovasc Dis 51:188-198 (2022). PubMed: 34515064
- Xu L et al. Tanshinone IIA regulates the TGF-β1/Smad signaling pathway to ameliorate non-alcoholic steatohepatitis-related fibrosis. Exp Ther Med 24:486 (2022). PubMed: 35761808
- Zhao B et al. Activation of TRPV4 by lactate as a critical mediator of renal fibrosis in spontaneously hypertensive rats after moderate- and high-intensity exercise. Front Physiol 13:927078 (2022). PubMed: 36160854
- You Y et al. Enhanced Expression of ARK5 in Hepatic Stellate Cell and Hepatocyte Synergistically Promote Liver Fibrosis. Int J Mol Sci 23:N/A (2022). PubMed: 36361872
- Yang J et al. CRTAC1 (Cartilage acidic protein 1) inhibits cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) process in bladder cancer by downregulating Yin Yang 1 (YY1) to inactivate the TGF-β pathway. Bioengineered 12:9377-9389 (2021). PubMed: 34818994