Recombinant Anti-Smad4 antibody [EPR22589-112] (ab230815)
![Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815)](https://www.abcam.com/ps/products/230/ab230815/Images/ab230815-5-230815WB1.jpg "Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815)")
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR22589-112] to Smad4
- Suitable for: IP, Flow Cyt (Intra), WB, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-Smad4 antibody [EPR22589-112]
See all Smad4 primary antibodies -
Description
Rabbit monoclonal [EPR22589-112] to Smad4 -
Host species
Rabbit -
Tested applications
Suitable for: IP, Flow Cyt (Intra), WB, ICC/IFmore details
Unsuitable for: ChIP or IHC-P -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HAP1, HeLa, HCT116, HepG2, RAW264.7, HEK-293 and NIH/3T3 whole cell lysates; human fetal lung, mouse embryo, mouse lung and rat lung tissue lysates. Flow Cyt (intra): WT HAP1 cells. ICC/IF: HeLa cell line treated with TGF-beta. IP: HeLa and NIH/3T3 whole cell lysates.
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General notes
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.
Properties
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Form
Liquid -
Storage instructions
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. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading... -
Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR22589-112 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab230815 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| IP |
1/30.
|
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| Flow Cyt (Intra) |
1/500.
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| WB |
1/1000. Detects a band of approximately 60 kDa (predicted molecular weight: 60 kDa).
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| ICC/IF |
1/100.
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| Notes |
|---|
|
IP
1/30. |
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Flow Cyt (Intra)
1/500. |
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WB
1/1000. Detects a band of approximately 60 kDa (predicted molecular weight: 60 kDa). |
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ICC/IF
1/100. |
Target
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Function
Common SMAD (co-SMAD) is the coactivator and mediator of signal transduction by TGF-beta (transforming growth factor). Component of the heterotrimeric SMAD2/SMAD3-SMAD4 complex that forms in the nucleus and is required for the TGF-mediated signaling. Promotes binding of the SMAD2/SMAD4/FAST-1 complex to DNA and provides an activation function required for SMAD1 or SMAD2 to stimulate transcription. Component of the multimeric SMAD3/SMAD4/JUN/FOS complex which forms at the AP1 promoter site; required for syngernistic transcriptional activity in response to TGF-beta. May act as a tumor suppressor. -
Involvement in disease
Defects in SMAD4 are a cause of pancreatic cancer (PNCA) [MIM:260350].
Defects in SMAD4 are a cause of juvenile polyposis syndrome (JPS) [MIM:174900]; also known as juvenile intestinal polyposis (JIP). JPS is an autosomal dominant gastrointestinal hamartomatous polyposis syndrome in which patients are at risk for developing gastrointestinal cancers. The lesions are typified by a smooth histological appearance, predominant stroma, cystic spaces and lack of a smooth muscle core. Multiple juvenile polyps usually occur in a number of Mendelian disorders. Sometimes, these polyps occur without associated features as in JPS; here, polyps tend to occur in the large bowel and are associated with an increased risk of colon and other gastrointestinal cancers.
Defects in SMAD4 are a cause of juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome (JP/HHT) [MIM:175050]. JP/HHT syndrome phenotype consists of the coexistence of juvenile polyposis (JIP) and hereditary hemorrhagic telangiectasia (HHT) [MIM:187300] in a single individual. JIP and HHT are autosomal dominant disorders with distinct and non-overlapping clinical features. The former, an inherited gastrointestinal malignancy predisposition, is caused by mutations in SMAD4 or BMPR1A, and the latter is a vascular malformation disorder caused by mutations in ENG or ACVRL1. All four genes encode proteins involved in the transforming-growth-factor-signaling pathway. Although there are reports of patients and families with phenotypes of both disorders combined, the genetic etiology of this association is unknown.
Defects in SMAD4 may be a cause of colorectal cancer (CRC) [MIM:114500]. -
Sequence similarities
Belongs to the dwarfin/SMAD family.
Contains 1 MH1 (MAD homology 1) domain.
Contains 1 MH2 (MAD homology 2) domain. -
Domain
The MH1 domain is required for DNA binding.
The MH2 domain is required for both homomeric and heteromeric interactions and for transcriptional regulation. Sufficient for nuclear import. -
Post-translational
modificationsMonoubiquitinated on Lys-519 by E3 ubiquitin-protein ligase TRIM33. Monoubiquitination hampers its ability to form a stable complex with activated SMAD2/3 resulting in inhibition of TGF-beta/BMP signaling cascade. Deubiqitination by USP9X restores its competence to mediate TGF-beta signaling. -
Cellular localization
Cytoplasm. Nucleus. Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with R-SMAD. - Information by UniProt
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Database links
- Entrez Gene: 4089 Human
- Entrez Gene: 17128 Mouse
- Entrez Gene: 50554 Rat
- Omim: 600993 Human
- SwissProt: Q13485 Human
- SwissProt: P97471 Mouse
- SwissProt: O70437 Rat
- Unigene: 75862 Human
see all -
Alternative names
- (Small) Mothers Against Decapentaplegic antibody
- Deleted in Pancreatic Carcinoma 4 antibody
- Deleted in Pancreatic Carcinoma antibody
see all
Images
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Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815)All lanes : Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution
Lane 1 : Smad4 knockout HAP1 whole cell lysate
Lane 2 : Wild-type HAP1 whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 5 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 6 : Human fetal lung tissue lysate
Lane 7 : Mouse embryo tissue lysate
Lane 8 : Mouse lung tissue lysate
Lane 9 : Rat lung tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 60 kDa
Observed band size: 60 kDaBlocking/Dilution buffer: 5% NFDM/TBST.
Exposure Time: Lanes 1-6: 15 secs; Lanes 7-8: 114 secs; Lane 9: 3 mins.
ab230815 was shown to specifically react with Smad4 in wild-type HAP1 cells as signal was lost in Smad4 knockout cells. Wild-type and Smad4 knockout samples were subjected to SDS-PAGE. ab230815 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.
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![Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (ab230815)](https://www.abcam.com/ps/products/230/ab230815/Images/ab230815-3-230815IP1.jpg)
Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (ab230815)Smad4 was immunoprecipitated from 0.35mg of HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab230815 at 1/30 dilution. Western Blot was performed from the immunoprecipitate using ab230815 at 1/1000 dilution (0.5 μg/ml).
VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.
Lane 1: HeLa whole cell lysate 10μg (Input).
Lane 2: ab230815 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab230815 in HeLa whole cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 30 seconds
Lysate was freshly prepared and IP test was done immediately to avoid protein degradation. Lysate incubation time was shortened from overnight to 2h.
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![Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [EPR22589-112] (ab230815)](https://www.abcam.com/ps/products/230/ab230815/Images/ab230815-1-230815ICC1.jpg)
Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [EPR22589-112] (ab230815)Immunofluorescent analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Smad4 with ab230815 at a 1/100 dilution (green). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Confocal image showing mainly nuclear staining in HeLa cell line treated with TGF-beta (10ng/ml) for 1 h. AlexaFluor®488 Goat anti-Rabbit (ab150077) was used secondary antibody at 1/1000 dilution, this was also used on its own as a control. DAPI was used as a nuclear counterstain (blue). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594, ab195889) was used as a counterstain at 1/200 dilution.
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![Flow Cytometry (Intracellular) - Anti-Smad4 antibody [EPR22589-112] (ab230815)](https://www.abcam.com/ps/products/230/ab230815/Images/ab230815-2-230815FC1.jpg)
Flow Cytometry (Intracellular) - Anti-Smad4 antibody [EPR22589-112] (ab230815)Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed Smad4 KO HAP1 (Smad4 knockout human chronic myelogenous leukemia near-haploid cell line, Left) / WT HAP1 (human chronic myelogenous leukemia near-haploid cell line, Right) cell line labeling Smad4 with ab230815 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor� 488, ab150077) was used as secondary antibody at 1/2000 dilution.
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![Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815)](https://www.abcam.com/ps/products/230/ab230815/Images/ab230815-6-230815WB2.jpg)
Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815)All lanes : Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution
Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 60 kDa
Exposure time: 48 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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![Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815)](https://www.abcam.com/ps/products/230/ab230815/Images/ab230815-7-230815WB3.jpg)
Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815)All lanes : Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution
Lane 1 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 60 kDa
Observed band size: 60 kDaBlocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: Lane 1: 15 secs; Lane 2: 7.75 secs.
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![Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (ab230815)](https://www.abcam.com/ps/products/230/ab230815/Images/ab230815-4-230815IP2.jpg)
Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (ab230815)Smad4 was immunoprecipitated from 0.35mg of NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab230815 at 1/30 dilution. Western Blot was performed from the immunoprecipitate using ab230815 at 1/1000 dilution (0.5 μg/ml).
VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.
Lane 1: NIH/3T3 whole cell lysate 10μg (Input).
Lane 2: ab230815 IP in NIH/3T3 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab230815 in NIH/3T3 whole cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 10 seconds
Lysate was freshly prepared and IP test was done immediately to avoid protein degradation. Lysate incubation time was shortened from overnight to 2h.
-
![Anti-Smad4 antibody [EPR22589-112] (ab230815)](https://www.abcam.com/ps/products/230/ab230815/Images/ab230815-8-benefits-of-recombinant-antibodies.png)
Anti-Smad4 antibody [EPR22589-112] (ab230815)
Protocols
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.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (2)
ab230815 has been referenced in 2 publications.
- Song Y et al. DEK-targeting aptamer DTA-64 attenuates bronchial EMT-mediated airway remodelling by suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathway in asthma. J Cell Mol Med 24:13739-13750 (2020). PubMed: 33124760
- Wang C et al. Cryptotanshinone Attenuates Airway Remodeling by Inhibiting Crosstalk Between Tumor Necrosis Factor-Like Weak Inducer of Apoptosis and Transforming Growth Factor Beta 1 Signaling Pathways in Asthma. Front Pharmacol 10:1338 (2019). PubMed: 31780948