Name: Anti-Smad3 antibody [EP568Y]
Brand: RabMAb
SKU: ab40854
Rating: 5 (9 reviews)

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Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EP568Y] to Smad3
Suitable for: Flow Cyt (Intra), WB, IHC-P, Sandwich ELISA, ICC/IF
Knockout validated
Reacts with: Rat, Human

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 594 Alexa Fluor® 647 Carrier Free HRP PE

产品名称

Anti-Smad3抗体[EP568Y]
参阅全部 Smad3 一抗
描述

兔单克隆抗体[EP568Y] to Smad3
宿主

Rabbit
经测试应用

适用于: Flow Cyt (Intra), WB, IHC-P, Sandwich ELISA, ICC/IFmore details
不适用于: IP
种属反应性

与反应: Rat, Human
预测可用于: Mouse, Chicken, Pig
免疫原

Synthetic peptide within Human Smad3 aa 200-300 (internal sequence). The exact sequence is proprietary.
Database link: P84022
(Peptide available as ab173094)
阳性对照
- WB: A549, HeLa, Human Kidney, HT-29, HT-1080 and Jurkat whole cell lysates; Rat liver tissue lysate. IHC-P: Human prostate carcinoma, breast carcinoma, colonic adenocarcinoma, lung adenocarcinoma, gastric adenocarcinoma, glioma and liver tissues. ICC/IF: HepG2 cells. Flow Cyt (intra): HCT116 and HT-29 cells.
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.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

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: 40% Glycerol, 0.05% BSA, 59% PBS
Concentration information loading...
纯度

Protein A purified
克隆

单克隆
克隆编号

EP568Y
同种型

IgG
研究领域

Alternative Versions
Compatible Secondaries
Conjugation kits
- PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
- APC Conjugation Kit - Lightning-Link® (ab201807)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
KO cell lines
- Human SMAD3 knockout A549 cell line (ab263915)
KO cell lysates
- Human SMAD3 knockout A549 cell lysate (ab264513)
Positive Controls
- HT-29 whole cell lysate (ab3952)
- Jurkat whole cell lysate (ab7899)
Recombinant Protein
- Recombinant Human Smad3 protein (ab89353)
Related Products
- Prestained Protein Ladder – Broad molecular weight (10-245 kDa) (ab116028)

The Abpromise guarantee

Abpromise™承诺保证使用ab40854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
Flow Cyt (Intra)		1/50 - 1/210. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
WB	(7)	1/1000 - 1/10000. Detects a band of approximately 55 kDa (predicted molecular weight: 48 kDa).
IHC-P	(1)	1/500 - 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. See IHC antigen retrieval protocols.
Sandwich ELISA		Use a concentration of 0.5 µg/ml. For sandwich ELISA, use this antibody as Detection at 0.5 µg/ml with Mouse monoclonal [AF9F7] to Smad3 (ab75512) as Capture.
ICC/IF	(1)	1/500 - 1/2000.

说明
Flow Cyt (Intra) 1/50 - 1/210. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
WB 1/1000 - 1/10000. Detects a band of approximately 55 kDa (predicted molecular weight: 48 kDa).
IHC-P 1/500 - 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. See IHC antigen retrieval protocols.
Sandwich ELISA Use a concentration of 0.5 µg/ml. For sandwich ELISA, use this antibody as Detection at 0.5 µg/ml with Mouse monoclonal [AF9F7] to Smad3 (ab75512) as Capture.
ICC/IF 1/500 - 1/2000.

应用说明

Is unsuitable for IP.

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 SMAD3/SMAD4 complex, activates transcription. Also can form a SMAD3/SMAD4/JUN/FOS complex at the AP-1/SMAD site to regulate TGF-beta-mediated transcription. Has an inhibitory effect on wound healing probably by modulating both growth and migration of primary keratinocytes and by altering the TGF-mediated chemotaxis of monocytes. This effect on wound healing appears to be hormone-sensitive. Regulator of chondrogenesis and osteogenesis and inhibits early healing of bone fractures. Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator.
疾病相关

Colorectal cancer
Loeys-Dietz syndrome 3
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.
结构域

The MH1 domain is required for DNA binding. Also binds zinc ions which are necessary for the DNA binding.
The MH2 domain is required for both homomeric and heteromeric interactions and for transcriptional regulation. Sufficient for nuclear import.
The linker region is required for the TGFbeta-mediated transcriptional activity and acts synergistically with the MH2 domain.
翻译后修饰

Phosphorylated on serine and threonine residues. Enhanced phosphorylation in the linker region on Thr-179, Ser-204 and Ser-208 on EGF and TGF-beta treatment. Ser-208 is the main site of MAPK-mediated phosphorylation. CDK-mediated phosphorylation occurs in a cell-cycle dependent manner and inhibits both the transcriptional activity and antiproliferative functions of SMAD3. This phosphorylation is inhibited by flavopiridol. Maximum phosphorylation at the G(1)/S junction. Also phosphorylated on serine residues in the C-terminal SXS motif by TGFBR1 and ACVR1. TGFBR1-mediated phosphorylation at these C-terminal sites is required for interaction with SMAD4, nuclear location and transactivational activity, and appears to be a prerequisite for the TGF-beta mediated phosphorylation in the linker region. Dephosphorylated in the C-terminal SXS motif by PPM1A. This dephosphorylation disrupts the interaction with SMAD4, promotes nuclear export and terminates TGF-beta-mediated signaling. Phosphorylation at Ser-418 by CSNK1G2/CK1 promotes ligand-dependent ubiquitination and subsequent proteasome degradation, thus inhibiting SMAD3-mediated TGF-beta responses. Phosphorylated by PDPK1.
Acetylation in the nucleus by EP300 in the MH2 domain regulates positively its transcriptional activity and is enhanced by TGF-beta.
Ubiquitinated. Monoubiquitinated, leading to prevent DNA-binding. Deubiquitination by USP15 alleviates inhibition and promotes activation of TGF-beta target genes.
Poly-ADP-ribosylated by PARP1 and PARP2. ADP-ribosylation negatively regulates SMAD3 transcriptional responses during the course of TGF-beta signaling.
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 (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).
Target information above from: UniProt accession P84022 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
数据库链接
- Entrez Gene: 4088 Human
- Entrez Gene: 17127 Mouse
- Entrez Gene: 397260 Pig
- Entrez Gene: 25631 Rat
- Omim: 603109 Human
- SwissProt: P84023 Chicken
- SwissProt: P84022 Human
- SwissProt: Q8BUN5 Mouse
- SwissProt: P84024 Pig
- SwissProt: P84025 Rat
- Unigene: 727986 Human
- Unigene: 742270 Human
- Unigene: 7320 Mouse
- Unigene: 10636 Rat
see all
别名
- DKFZP586N0721 antibody
- DKFZp686J10186 antibody
- hMAD 3 antibody
- hMAD-3 antibody
- hSMAD3 antibody
- HSPC193 antibody
- HST17436 antibody
- JV15 2 antibody
- JV15-2 antibody
- JV152 antibody
- LDS1C antibody
- LDS3 antibody
- MAD (mothers against decapentaplegic Drosophila) homolog 3 antibody
- MAD homolog 3 antibody
- Mad homolog JV15 2 antibody
- Mad protein homolog antibody
- MAD, mothers against decapentaplegic homolog 3 antibody
- Mad3 antibody
- MADH 3 antibody
- MADH3 antibody
- MGC60396 antibody
- Mothers against decapentaplegic homolog 3 antibody
- Mothers against DPP homolog 3 antibody
- SMA and MAD related protein 3 antibody
- SMAD 3 antibody
- SMAD antibody
- SMAD family member 3 antibody
- SMAD, mothers against DPP homolog 3 antibody
- Smad3 antibody
- SMAD3_HUMAN antibody
see all

Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

All lanes : Anti-Smad3 antibody [EP568Y] (ab40854) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : SMAD3 knockout A549 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Human Kidney cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 48 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab40854 observed at 48 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab40854 was shown to react with Smad3 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab40854 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

Immunohistochemical analysis of paraffin-embedded human colonic adenocarcinoma tissue labelling Smad3 with unpurified ab40854.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunocytochemistry/ Immunofluorescence - Anti-Smad3 antibody [EP568Y] (ab40854)

Immunocytochemsitry/Immunofluorescence analysis of HepG2 cells labelling Smad3 (green) with purified ab40854 at 1/2000 . Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody (green, left panel). Counterstained with DAPI (blue, right panel).
Flow Cytometry (Intracellular) - Anti-Smad3 antibody [EP568Y] (ab40854)

Overlay histogram showing HCT116 cells stained with unpurifiedab40854 (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 (ab40854, 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 IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
Sandwich ELISA - Anti-Smad3 antibody [EP568Y] (ab40854)

Standard Curve for Smad3 (Analyte: Smad3 protein (His tag) (ab89353, unpurified)); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [AF9F7] to Smad3 (ab75512) at 5µg/ml and Detector Antibody Rabbit monoclonal [EP568Y] to Smad3 (ab40854) at 0.5µg/ml.
Immunocytochemistry/ Immunofluorescence - Anti-Smad3 antibody [EP568Y] (ab40854)This image is courtesy of an Abreview submitted by Francesco Elia Marino

ab40854 staining Smad3 in human granulosa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with ethanol and triton and blocked for 1 hour at 26°C. Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. An undiluted IRDye^® 800CW-conjugated goat anti-rabbit IgG (H+L) polyclonal was used as the secondary antibody. Left - negative control (4 replicates).
See Abreview
Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

All lanes : Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5300 dilution (purified)

Lane 1 : HT-29 cell lysate
Lane 2 : HT-1080 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 48 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5000 dilution (unpurified) + Jurkat cell lysate at 10 µg

Predicted band size: 48 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?
Western blot - Anti-Smad3 antibody [EP568Y] (ab40854)

Anti-Smad3 antibody [EP568Y] (ab40854) at 1/5300 dilution (purified) + Rat liver tissue lysate at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 48 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
Flow Cytometry (Intracellular) - Anti-Smad3 antibody [EP568Y] (ab40854)

Intracellular Flow Cytometry analysis of HT-29 cells labelling Smad3 with purified ab40854 at 1/210 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Green - Isotype control, rabbit monoclonal IgG.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Smad3 with purified ab40854 at 1/2000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostate carcinoma tissue labelling unpurified ab40854 at 1/100 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling unpurified ab40854.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung adenocarcinoma tissue labelling unpurified ab40854.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue labelling unpurified ab40854.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody [EP568Y] (ab40854)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric adenocarcinoma tissue labelling unpurified ab40854.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Anti-Smad3 antibody [EP568Y] (ab40854)

Protocol Booklet

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发表研究结果有使用 ab40854？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab40854 被引用在 222 文献中.

Ren C et al. SHCBP1 Promotes the Progression of Esophageal Squamous Cell Carcinoma Via the TGFß Pathway. Appl Immunohistochem Mol Morphol 29:136-143 (2021). PubMed: 32769441
Wang H et al. Isolation and identification of goose skeletal muscle satellite cells and preliminary study on the function of C1q and tumor necrosis factor-related protein 3 gene. Anim Biosci 34:1078-1087 (2021). PubMed: 33152229
Tamura Y et al. Anti-pyroptotic function of TGF-ß is suppressed by a synthetic dsRNA analogue in triple negative breast cancer cells. Mol Oncol 15:1289-1307 (2021). PubMed: 33342034
Wei X et al. Ultrasound-targeted microbubble destruction-mediated Galectin-7-siRNA promotes the homing of bone marrow mesenchymal stem cells to alleviate acute myocardial infarction in rats. Int J Mol Med 47:677-687 (2021). PubMed: 33416139
Gori I et al. Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-ß responses through SKI stabilization. Elife 10:N/A (2021). PubMed: 33416497

View all Publications for this product

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Application

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)

Sample

Human Tissue sections (Lung tissue)

Antigen retrieval step

None

Permeabilization

Specification

Lung tissue

Blocking step

Zytomed Systems HRP Polymer Blocking Reagent as blocking agent for 5 minute(s) · Concentration: 100% · Temperature: 25°C

Fixative

HOPE-fixation

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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提交于 Oct 06 2015

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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Key features and details

Related conjugates and formulations

概述

产品名称

描述

宿主

经测试应用

种属反应性

免疫原

阳性对照

常规说明

性能

形式

存放说明

存储溶液

纯度

克隆

克隆编号

同种型

研究领域

相关产品

Alternative Versions

Compatible Secondaries

Conjugation kits

Isotype control

KO cell lines

KO cell lysates

Positive Controls

Recombinant Protein

Related Products

应用

The Abpromise guarantee

靶标

功能

疾病相关

序列相似性

结构域

翻译后修饰

细胞定位

数据库链接

别名

图片

实验方案

数据表及文件

SDS download

Datasheet download

文献 (222)

客户评价及客户问答

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Smad3 antibody [EP568Y]