Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP618Y] to Smad4 (HRP)
- Suitable for: IHC-P, WB
- Knockout validated
- Reacts with: Human
- Conjugation: HRP
Product nameAnti-Smad4 antibody [EP618Y] (HRP)
See all Smad4 primary antibodies
DescriptionRabbit monoclonal [EP618Y] to Smad4 (HRP)
Tested applicationsSuitable for: IHC-P, WBmore details
Species reactivityReacts with: Human
Predicted to work with: Mouse, Rat
Synthetic peptide within Human Smad4 aa 500 to the C-terminus (C terminal). The exact sequence is proprietary.
- WB: SH SY5Y whole cell lysate. IHC-P: normal human colon tissue.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. Store In the Dark.
Storage bufferpH: 7.40
Preservative: 0.1% 10% Proclin 300 Solution
Constituents: 30% Glycerol, 1% BSA, PBS
Concentration information loading...
PurityProtein A purified
Our Abpromise guarantee covers the use of ab195554 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|WB||1/5000. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).|
FunctionCommon 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 diseaseDefects 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 similaritiesBelongs to the dwarfin/SMAD family.
Contains 1 MH1 (MAD homology 1) domain.
Contains 1 MH2 (MAD homology 2) domain.
DomainThe 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.
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 localizationCytoplasm. Nucleus. Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with R-SMAD.
- Information by UniProt
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All lanes : Anti-Smad4 antibody [EP618Y] (HRP) (ab195554) at 1/5000 dilution
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : SMAD4 knockout HAP1 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 65 kDa
Observed band size: 65 kDa
Exposure time: 12 minutes
ab195554 was shown to recognize Smad4 in wild-type HAP1 cells as signal was lost at the expected MW in SMAD4 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SMAD4 knockout samples were subjected to SDS-PAGE. Ab195554 and ab184095 (Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control (Alexa Fluor® 680) loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.
Anti-Smad4 antibody [EP618Y] (HRP) (ab195554) at 1/5000 dilution + SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 65 kDa
Observed band size: 65 kDa
Exposure time: 20 minutes
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab195554 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.
IHC image of Smad4 staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*, performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab195554 at 1/100 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
ab195554 has not yet been referenced specifically in any publications.