Anti-Smad3 antibody (ab84177)
Key features and details
- Rabbit polyclonal to Smad3
- Suitable for: IP, ICC/IF, WB, IHC-P
- Knockout validated
- Reacts with: Mouse, Human
- Isotype: IgG
Get better batch-to-batch reproducibility with a recombinant antibody
- Research with confidence – consistent and reproducible results with every batch
- Long-term and scalable supply – powered by recombinant technology for fast production
- Success from the first experiment – confirmed specificity through extensive validation
- Ethical standards compliant – production is animal-free
Overview
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Product name
Anti-Smad3 antibody
See all Smad3 primary antibodies -
Description
Rabbit polyclonal to Smad3 -
Host species
Rabbit -
Tested applications
Suitable for: IP, ICC/IF, WB, IHC-Pmore details -
Species reactivity
Reacts with: Mouse, Human
Predicted to work with: Rat, Sheep, Horse, Chicken, Pig
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Immunogen
Synthetic peptide corresponding to Human Smad3 aa 150-250 conjugated to keyhole limpet haemocyanin.
(Peptide available asab95046) -
Positive control
- This antibody gave a positive signal in HeLa whole cell lysate; A549 cell lysate, HeLa nuclear extract and EGF-treated A431 whole cell lysate.
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General notes
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.
If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As
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 or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help. -
Concentration information loading... -
Purity
Immunogen affinity purified -
Clonality
Polyclonal -
Isotype
IgG -
Research areas
Associated products
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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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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab84177 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 |
Use at an assay dependent concentration.
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| ICC/IF |
Use a concentration of 5 µg/ml.
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| WB | (1) |
Use a concentration of 1 µg/ml. Detects a band of approximately 52 kDa (predicted molecular weight: 48 kDa).
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| IHC-P |
Use a concentration of 1 µg/ml.
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| Notes |
|---|
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IP
Use at an assay dependent concentration. |
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ICC/IF
Use a concentration of 5 µg/ml. |
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WB
Use a concentration of 1 µg/ml. Detects a band of approximately 52 kDa (predicted molecular weight: 48 kDa). |
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IHC-P
Use a concentration of 1 µg/ml. |
Target
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Function
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. -
Involvement in disease
Colorectal cancer
Loeys-Dietz syndrome 3 -
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. 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. -
Post-translational
modificationsPhosphorylated 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. -
Cellular localization
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). - Information by UniProt
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Database links
- 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
see all -
Alternative names
- DKFZP586N0721 antibody
- DKFZp686J10186 antibody
- hMAD 3 antibody
see all
Images
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Western blot - Anti-Smad3 antibody (ab84177)All lanes : Anti-Smad3 antibody (ab84177) at 1 µg/ml
Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : SMAD3 knockout A549 (Human lung carcinoma cell line) whole 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 - 2: Merged signal (red and green). Green - ab84177 observed at 50 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab84177 was shown to react with Smad3 in wild-type A549 cells in western blot with loss of signal observed in SMAD3 knockout sample. Wild-type and SMAD3 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab84177 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml 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.
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Immunoprecipitation - Anti-Smad3 antibody (ab84177)Smad3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Smad3 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab84177.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 50kDa: Smad3 -
Immunocytochemistry/ Immunofluorescence - Anti-Smad3 antibody (ab84177)ICC/IF image of ab84177 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab84177, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa cells at 5µg/ml. -
Western blot - Anti-Smad3 antibody (ab84177)All lanes : Anti-Smad3 antibody (ab84177) at 1 µg/ml
Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : EGF-treated A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg/ml per lane.
Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 48 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?
Exposure time: 20 minutes -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad3 antibody (ab84177)IHC image of Ab84177 staining in Human Cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with Ab84177, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
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. -
Western blot - Anti-Smad3 antibody (ab84177)All lanes : Anti-Smad3 antibody (ab84177) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 48 kDa
Observed band size: 48 kDa
Additional bands at: 37 kDa, 50 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 8 minutes
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (13)
ab84177 has been referenced in 13 publications.
- 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
- Wang Y et al. Metformin Ameliorates Chronic Colitis-Related Intestinal Fibrosis via Inhibiting TGF-β1/Smad3 Signaling. Front Pharmacol 13:887497 (2022). PubMed: 35645830
- Carr D et al. Multiple roles for Bcl-3 in mammary gland branching, stromal collagen invasion, involution and tumor pathology. Breast Cancer Res 24:40 (2022). PubMed: 35681213
- Shih CC et al. Protective Effects of One 2,4-Dihydro-3H-Pyrazol-3-one Derivative against Posterior Capsular Opacification by Regulation of TGF-β2/SMADs and Non-SMAD Signaling, Collagen I, and Fibronectin Proteins. Curr Issues Mol Biol 44:5048-5066 (2022). PubMed: 36286058
- Allen J Effects of corticosteroids vs halofuginone on vocal fold wound healing in an ovine model. Laryngoscope Investig Otolaryngol 6:786-793 (2021). PubMed: 34401503