Recombinant Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR23681-40] to Smad2 (phospho S467)
- Suitable for: IHC-P, Flow Cyt (Intra), Dot blot, WB, IP, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
-
Product name
Anti-Smad2 (phospho S467) antibody [EPR23681-40]
See all Smad2 primary antibodies -
Description
Rabbit monoclonal [EPR23681-40] to Smad2 (phospho S467) -
Host species
Rabbit -
Specificity
ab280888 was raised against a dual phosphorated peptide for both phosopho S467 and phosopho S465. The dot blot shows that ab280888 binds phosopho S467 on its own, but binding is enhanced by the presence of phosopho S465. Our data suggests shown in the dot blot that ab280888 does not bind phosopho S465 alone.
-
Tested applications
Suitable for: IHC-P, Flow Cyt (Intra), Dot blot, WB, IP, ICC/IFmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
-
Positive control
- WB: Wild-type HeLa treated/untreated with 20 ng/ml TGF beta1. NIH/3T3 treated/untreated with 20ng/ml TGF beta1 and 50 µM MG-132. IHC-P: Human stomach tissue. Human colon carcinoma tissue. Mouse cardiac muscle. Rat lung tissue. ICC/IF: HeLa and NIH/3T3 cells treated with TGF beta1. Flow Cyt (intra): HeLa and NIH/3T3 cells treated with TGF beta1. IP: HeLa and NIH/3T3 cells treated with TGF beta1.
-
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
-
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
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
-
Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR23681-40 -
Isotype
IgG -
Research areas
Associated products
-
Alternative Versions
-
Compatible Secondaries
-
Isotype control
-
KO cell lines
-
KO cell lysates
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab280888 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 |
---|---|---|
IHC-P |
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
|
|
Flow Cyt (Intra) |
1/5000.
|
|
Dot blot |
1/1000.
|
|
WB |
1/1000. Predicted molecular weight: 52 kDa.
|
|
IP |
1/30.
|
|
ICC/IF |
1/50.
|
Notes |
---|
IHC-P
1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Flow Cyt (Intra)
1/5000. |
Dot blot
1/1000. |
WB
1/1000. Predicted molecular weight: 52 kDa. |
IP
1/30. |
ICC/IF
1/50. |
Target
-
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 SMAD2/SMAD4 complex, activates transcription. May act as a tumor suppressor in colorectal carcinoma. -
Tissue specificity
Expressed at high levels in skeletal muscle, heart and placenta. -
Sequence similarities
Belongs to the dwarfin/SMAD family.
Contains 1 MH1 (MAD homology 1) domain.
Contains 1 MH2 (MAD homology 2) domain. -
Post-translational
modificationsPhosphorylated 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. -
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. On dephosphorylation by phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1. - Information by UniProt
-
Database links
- Entrez Gene: 4087 Human
- Entrez Gene: 17126 Mouse
- Entrez Gene: 29357 Rat
- Omim: 601366 Human
- SwissProt: Q15796 Human
- SwissProt: Q62432 Mouse
- SwissProt: O70436 Rat
- Unigene: 12253 Human
see all -
Alternative names
- Drosophila, homolog of, MADR2 antibody
- hMAD-2 antibody
- HsMAD2 antibody
see all
Images
-
All lanes : Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution
Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate
Lane 3 : Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/20000 dilution
Predicted band size: 52 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lanes 1-4: Merged signal (red and green). Green - ab280888 observed at 60 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.
ab280888 Anti-Smad2 (phospho S467) antibody [EPR23681-40] was shown to specifically react with Smad2 in wild-type HeLa cells. 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.
ab280888 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4? overnight at 1 in 1000 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 10000 dilution for 1 hour at room temperature before imaging.
-
Dot blot analysis of Smad2 (phospho S467) using ab280888 at 1:1000 (0.524 µg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.
Lane 1: Smad2 peptide (aa 458-467)
Lane 2: Smad2 (phospho S465) peptide (aa 458-467)
Lane 3: Smad2 (phospho aa S465/S467) peptide (aa 458-467)
Lane 4: Smad2 (phospho S467) peptide (aa 458-467)Exposure time: 3 minutes
Blocking and diluting buffer and concentration: 5% NFDM/TBST
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunohistochemical analysis of paraffin-embedded human stomach tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human stomach without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
-
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labelling Smad2 (phospho S467) with ab280888 at 1/50 (10.48 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).
Confocal image showing mainly nuclear staining in HeLa cells treated with TGF-β1 (20 ng/ml) for 15 mins.
ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
-
Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes (Red)/ Untreated control (Green) cells labelling Smad2 (phospho S467) with ab280888 at 1/5000 dilution (0.01µg)/ red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in HeLa (PMID:24959295).
-
Smad2 (phospho S467) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate 10 µg with ab280888 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280888 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: HeLa treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate 10 µg
Lane 2: ab280888 IP in HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280888 in HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 15 seconds.
-
All lanes : Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution
Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate at 10 µg
Lane 2 : HeLa treated with 20ng/ml TGF beta1 for 15 minutes whole cell lysate (phosphatase treated membrane) at 10 µg
Lane 3 : HeLa whole cell lysate at 20 µg
Lane 4 : HeLa treated with 20ng/ml TGF beta1 for 15 minutes, whole cell lysate at 20 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 52 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST
The molecular weight observed is consistent with what has been described in the literature. (PMID: 24959295)
The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in HeLa (PMID:24959295).Exposure time: 3 minutes
-
All lanes : Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888) at 1/1000 dilution
Lane 1 : NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 and 50 µM MG-132 for 15 minutes, whole cell lysate
Lane 2 : NIH/3T3 treated with 20ng/ml TGF beta1 and 50 µM MG-132 for 15 minutes whole cell lysate (phosphatase treated membrane)
Lane 3 : NIH/3T3 whole cell lysate
Lane 4 : NIH/3T3 treated with 20ng/ml TGF beta1 and 50 µM MG-132 for 15 minutes, whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 52 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST
The molecular weight observed is consistent with what has been described in the literature. (PMID: 24347165)
The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in NIH/3T3 (PMID: 24347165)
Exposure time: 3 minutes
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 µg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human colon carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunohistochemical analysis of paraffin-embedded mouse cardiac muscle tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 µg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse cardiac muscle without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunohistochemical analysis of paraffin-embedded rat lung tissue labelling Smad2 (phospho S467) with ab280888 at 1/2000 (0.262 µg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat lung without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab280888 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
-
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 (phospho S467) antibody [EPR23681-40] (ab280888)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labelling Smad2 (phospho S467) with ab280888 at 1/50 (10.48 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).
Confocal image showing mainly nuclear staining in NIH/3T3 cells treated with TGF-β1 (20 ng/ml) for 15 mins.
ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
-
Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 for 15 minutes (Red)/ Untreated control (Green) cells labelling Smad2 (phospho S467) with ab280888 at 1/5000 dilution (0.01µg)/ red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
The up-regulation of pSmad2 (S467) is induced by TGF-beta1 treatment in NIH/3T3 (PMID: 24347165).
-
Smad2 (phospho S467) was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) treated with 20ng/ml TGF beta1 50 µM MG-132 for 15 minutes, whole cell lysate 10 µg with ab280888 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280888 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: NIH/3T3 treated with 20ng/ml TGF beta1 50 µM MG-132 for 15 minutes, whole cell lysate 10 µg
Lane 2: ab280888 IP in NIH/3T3 treated with 20ng/mltreated with 20ng/ml TGF beta1 50 µM MG-132 for 15 minutes, whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280888 in NIH/3T3 treated with 20ng/mltreated with 20ng/ml TGF beta1 50 µM MG-132 for 15 minutes, whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 32 seconds.
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
-
SDS download
-
Datasheet download
Certificate of Compliance
References (12)
ab280888 has been referenced in 12 publications.
- 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
- 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. HERC3 regulates epithelial-mesenchymal transition by directly ubiquitination degradation EIF5A2 and inhibits metastasis of colorectal cancer. Cell Death Dis 13:74 (2022). PubMed: 35064108
- Li G et al. miR-155 inhibits chondrocyte pyroptosis in knee osteoarthritis by targeting SMAD2 and inhibiting the NLRP3/Caspase-1 pathway. J Orthop Surg Res 17:48 (2022). PubMed: 35090521
- Jin D et al. Impact of klotho on the expression of SRGAP2a in podocytes in diabetic nephropathy. BMC Nephrol 23:151 (2022). PubMed: 35436879