重组Anti-Smad2抗体[EP567Y] (ab33875)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP567Y] to Smad2
- Suitable for: Flow Cyt (Intra), ChIC/CUT&RUN-seq, WB, ICC/IF
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
概述
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产品名称
Anti-Smad2抗体[EP567Y]
参阅全部 Smad2 一抗 -
描述
兔单克隆抗体[EP567Y] to Smad2 -
宿主
Rabbit -
特异性
This antibody detects a region about 40AA before the MH2 region (not the MH2 region itself). -
经测试应用
适用于: Flow Cyt (Intra), ChIC/CUT&RUN-seq, WB, ICC/IFmore details
不适用于: IHC-P or IP -
种属反应性
与反应: Mouse, Human
预测可用于: Rat -
免疫原
Synthetic peptide within Human Smad2 aa 200-300. The exact sequence is proprietary.
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阳性对照
- WB: HeLa, A549, RAW264.7, and Jurkat cell lysate ICC/IF: HeLa cells Flow Cyt (intra): PC3 and Jurkat cells ChIC/CUT&RUN seq: HaCaT cell.
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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. Avoid freeze / thaw cycle. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% 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
EP567Y -
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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Positive Controls
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Recombinant Protein
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab33875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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Flow Cyt (Intra) |
1/110.
For unpurified, use 1/70. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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ChIC/CUT&RUN-seq |
Use at an assay dependent concentration.
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WB | (2) |
1/1000 - 1/2000. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa).
For unpurified, use 1/1000. |
ICC/IF |
1/300.
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说明 |
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Flow Cyt (Intra)
1/110. For unpurified, use 1/70. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
ChIC/CUT&RUN-seq
Use at an assay dependent concentration. |
WB
1/1000 - 1/2000. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa). For unpurified, use 1/1000. |
ICC/IF
1/300. |
靶标
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功能
Receptor-regulated SMAD (R-SMAD) that is an intracellular signal transducer and transcriptional modulator activated by TGF-beta (transforming growth factor) and activin type 1 receptor kinases. Binds the TRE element in the promoter region of many genes that are regulated by TGF-beta and, on formation of the SMAD2/SMAD4 complex, activates transcription. May act as a tumor suppressor in colorectal carcinoma. -
组织特异性
Expressed at high levels in skeletal muscle, heart and placenta. -
序列相似性
Belongs to the dwarfin/SMAD family.
Contains 1 MH1 (MAD homology 1) domain.
Contains 1 MH2 (MAD homology 2) domain. -
翻译后修饰
Phosphorylated on one or several of Thr-220, Ser-245, Ser-250, and Ser-255. In response to TGF-beta, phosphorylated on Ser-465/467 by TGF-beta and activin type 1 receptor kinases. Able to interact with SMURF2 when phosphorylated on Ser-465/467, recruiting other proteins, such as SNON, for degradation. In response to decorin, the naturally occurring inhibitor of TGF-beta signaling, phosphorylated on Ser-240 by CaMK2. Phosphorylated by MAPK3 upon EGF stimulation; which increases transcriptional activity and stability, and is blocked by calmodulin.
In response to TGF-beta, ubiquitinated by NEDD4L; which promotes its degradation.
Acetylated on Lys-19 by coactivators in response to TGF-beta signaling, which increases transcriptional activity. Isoform short: Acetylation increases DNA binding activity in vitro and enhances its association with target promoters in vivo. Acetylation in the nucleus by EP300 is enhanced by TGF-beta. -
细胞定位
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. - Information by UniProt
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数据库链接
- Entrez Gene: 4087 Human
- Entrez Gene: 17126 Mouse
- Entrez Gene: 29357 Rat
- Omim: 601366 Human
- SwissProt: Q15796 Human
- SwissProt: Q62432 Mouse
- SwissProt: O70436 Rat
- Unigene: 12253 Human
see all -
别名
- Drosophila, homolog of, MADR2 antibody
- hMAD-2 antibody
- HsMAD2 antibody
see all
图片
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ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 2.5 x 10^5 HaCaT (Human keratinocyte cell line) cells (treated with 7ng/ml TGF-β for 1h) and 5 µg of ab33875 [EP567Y]. 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. -
All lanes : Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : SMAD2 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 58 kDaLanes 1 - 4: Merged signal (red and green). Green - ab33875 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab33875 was shown to react with Smad2 in wild-type HeLa. Loss of signal was observed when knockout cell line ab255430 (knockout cell lysate ab263833) was used. Wild-type and Smad2 knockout samples were subjected to SDS-PAGE. ab33875 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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.
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All lanes : Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution
Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : SMAD2 knockout HeLa whole cell lysate
Lane 3 : A549 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 58 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?Lanes 1 - 3: Merged signal (red and green). Green - ab33875 observed at 52 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab33875 was shown to specifically react with Smad2 in wild-type WT HeLa cells as signal was lost in SMAD2 knockout cells. Wild-type and SMAD2 knockout samples were subjected to SDS-PAGE. Ab33875 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.
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Immunocytochemistry/Immunofluorescent analysis of HeLa (human cervix adenocarcinoma epithelial) cells labeling Smad2 with ab33875 at a dilution of 1/500. ab150077, an Alexa Fluor® 488 goat anti-rabbit was used at 1/1000 was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Counterstain antibody: ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200.
Secondary antibody only negative control is shown in the bottom panels.
Confocal image showing mainly nuclear staining on HeLa cells after the treatment with TGF-b (10ng/mL) for 1 hour.
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Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution (Purified) + RAW264.7 at 10 µg
Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 58 kDa
Observed band size: 58 kDaBlocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
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Anti-Smad2 antibody [EP567Y] (ab33875) at 1/2000 dilution (Purified) + Jurkat cell lysate at 10 µg
Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 58 kDa
Observed band size: 58 kDaBlocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
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Overlay histogram showingJurkat cells stained with purified ab33875 (pink line) at a dilution of 1/110. The cells were fixed with 2% PFA.FITC goat anti-rabbit was used at a dilution of 1/150 and rabbit monoclonal IgG was used as theisotype control (green line).
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Anti-Smad2 antibody [EP567Y] (ab33875) at 1/500 dilution + RAW264.7 cell lysate at 10 µg
Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 58 kDa
Additional bands at: 58 kDa. We are unsure as to the identity of these extra bands.Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
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Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution (Unpurified) + Jurkat cell lysate at 10 µg
Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 58 kDa
Additional bands at: 58 kDa. We are unsure as to the identity of these extra bands.Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
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Overlay histogram showing PC3 cells stained with unpurified ab33875 (red line). The cells were fixed with methanol (5 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 (ab33875, 1/100 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 PC3 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
数据表及文件
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SDS download
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Datasheet download
文献 (54)
ab33875 被引用在 54 文献中.
- Sun B et al. Irisin reduces bone fracture by facilitating osteogenesis and antagonizing TGF-β/Smad signaling in a growing mouse model of osteogenesis imperfecta. J Orthop Translat 38:175-189 (2023). PubMed: 36439629
- Li S et al. Fibroblast growth factor-21 as a novel metabolic factor for regulating thrombotic homeostasis. Sci Rep 12:400 (2022). PubMed: 35013379
- Deng W et al. Silencing lncRNA Snhg6 mitigates bleomycin-induced pulmonary fibrosis in mice via miR-26a-5p/TGF-β1-smads axis. Environ Toxicol 37:2375-2387 (2022). PubMed: 35785413
- Tian H et al. Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration. iScience 25:105050 (2022). PubMed: 36185374
- Zhang Y et al. Pien-Tze-Huang alleviates CCl4-induced liver fibrosis through the inhibition of HSC autophagy and the TGF-β1/Smad2 pathway. Front Pharmacol 13:937484 (2022). PubMed: 36188553