重组Anti-Smad2抗体[EP784Y] (ab40855)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP784Y] to Smad2
- Suitable for: IHC-P, IP, ICC/IF, Flow Cyt (Intra), ChIC/CUT&RUN-seq, WB
- Knockout validated
- Reacts with: Rat, Human
Related conjugates and formulations
概述
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产品名称
Anti-Smad2抗体[EP784Y]
参阅全部 Smad2 一抗 -
描述
兔单克隆抗体[EP784Y] to Smad2 -
宿主
Rabbit -
特异性
This antibody is specific for MH 1 domain of Smad2. -
经测试应用
适用于: IHC-P, IP, ICC/IF, Flow Cyt (Intra), ChIC/CUT&RUN-seq, WBmore details -
种属反应性
与反应: Rat, Human -
免疫原
Synthetic peptide within Human Smad2 aa 50-150. The exact sequence is proprietary.
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阳性对照
- WB: A549, Jurkat, HeLa, A-673, HUVEC and C6 cell lysates. IP: HeLa IHC-P: Human bladder and prostate carcinoma tissue. ICC/IF: HeLa cells. Flow Cyt (intra): HeLa and PC3 cells. ChIC/CUT&RUN seq: HaCaT cell
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常规说明
The rat recommendation is based on the WB results. This antibody may not be suitable for IHC with rat samples.
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.
Mouse: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 40% Glycerol, 0.05% BSA, 59% PBS -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
EP784Y -
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ab40855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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IHC-P |
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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IP |
1/20 - 1/50.
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ICC/IF |
1/100 - 1/250.
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Flow Cyt (Intra) |
1/20 - 1/100.
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 | (4) |
1/2000 - 1/10000. Detects a band of approximately 55 kDa (predicted molecular weight: 58 kDa).
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说明 |
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IHC-P
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
IP
1/20 - 1/50. |
ICC/IF
1/100 - 1/250. |
Flow Cyt (Intra)
1/20 - 1/100. 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
1/2000 - 1/10000. Detects a band of approximately 55 kDa (predicted molecular weight: 58 kDa). |
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: 29357 Rat
- Omim: 601366 Human
- SwissProt: Q15796 Human
- SwissProt: O70436 Rat
- Unigene: 12253 Human
- Unigene: 705764 Human
- Unigene: 2755 Rat
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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 ab40855 [EP784Y]. 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 [EP784Y] (ab40855) at 1/2000 dilution
Lane 1 : 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.
Predicted band size: 58 kDaLanes 1 - 4: Merged signal (red and green). Green - ab40855 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab40855 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. ab40855 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.
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All lanes : Anti-Smad2 antibody [EP784Y] (ab40855) 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 kDaLanes 1 - 3: Merged signal (red and green). Green - ab40855 observed at 58 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab40855 was shown to specifically react with Smad2 in wild-type HeLa cells as signal was lost in Smad2 knockout cells. Wild-type and SMAD2 knockout samples were subjected to SDS-PAGE. Ab40855 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 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. -
All lanes : Anti-Smad2 antibody [EP784Y] (ab40855) at 1/2000 dilution
Lane 1 : A-673 (Human muscle Ewing's Sarcoma cell line) whole cell lysate
Lane 2 : HUVEC (Human umbilical vein endothelial cell line) whole cell lysate
Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 58 kDaDiluting and blocking buffer: 5% NFDM /TBST
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ab40855 staining Smad2 in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab195889 was used as a counterstain for primary antibody ab40855 at 1/1000. DAPI was used as a nuclear counterstain and PBS as a negative control.
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All lanes : Anti-Smad2 antibody [EP784Y] (ab40855) at 1/10000 dilution
Lane 1 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lanes 2-3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 20000 µg (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 58 kDaBlocking and diluting buffer: 5% NFDM/TBST
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ab40855 stainingSmad2 in human bladder carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/50. A ImmunoHistoProbe one step HRP Polymer was used as a secondary antibody, ready to use.
Negative control 1: PBS in place of primary antibody.
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ab40855 (purified) at 1/20 immunoprecipitating EGFR in HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate.
Lane 1 (input): HeLa whole cell lysate (10µg)
Lane 2 (+): ab40855 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40855 in HeLa whole cell lysate.
For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/1000).
Blocking/Diluting buffer and concentration: 5% NFDM/TBST.
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ab40855 staining Smad2in the human cell line HeLa (Human epithelial cell line from cervix adenocarcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde 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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Immunofluorescence staining of HeLa cells with purified ab40855 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was an Alexa Fluor® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.
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Overlay histogram showing PC3 cells stained with ab40855 (red line). The cells were fixed with 80% 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 (ab40855, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
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Anti-Smad2 antibody [EP784Y] (ab40855) at 1/500000 dilution + Jurkat cell lysate
Predicted band size: 58 kDa
Observed band size: 58 kDa -
ab40855 at a 1:100 dilution staining Smad2 in human prostate carcinoma tissue.
数据表及文件
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SDS download
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Datasheet download
文献 (89)
ab40855 被引用在 89 文献中.
- Zheng Y et al. Anti-PAI-1 Monoclonal Antibody Inhibits the Metastasis and Growth of Esophageal Squamous Cell Carcinoma. J Cancer 14:114-128 (2023). PubMed: 36605486
- Wang C et al. lncRNA XIST knockdown suppresses cell proliferation and promotes apoptosis in diabetic cataracts through the miR‑34a/SMAD2 axis. Mol Med Rep 25:N/A (2022). PubMed: 34751414
- Wei W et al. Feibi decoction-medicated serum inhibits lipopolysaccharide-induced inflammation in RAW264.7 cells and BMDMs. Exp Ther Med 23:110 (2022). PubMed: 34976152
- Wu F et al. TGF-βRII regulates glucose metabolism in oral cancer-associated fibroblasts via promoting PKM2 nuclear translocation. Cell Death Discov 8:3 (2022). PubMed: 35013150
- Zhang Z et al. Circ_0002623 promotes bladder cancer progression by regulating the miR-1276/SMAD2 axis. Cancer Sci 113:1250-1263 (2022). PubMed: 35048477