Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)

Rabbit Recombinant Monoclonal SMAD2 antibody. Suitable for ChIC/CUT&RUN-seq, IP, ChIP, WB, ICC/IF, Flow Cyt (Intra) and reacts with Human, Recombinant fragment - Human samples. Cited in 7 publications.

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Images

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (AB207447), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	ChIC/CUT&RUN-seq	IP	ChIP	WB	ICC/IF	Flow Cyt (Intra)
Human	Tested	Tested	Tested	Tested	Tested	Tested
Recombinant fragment - Human	Not recommended	Not recommended	Not recommended	Tested	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/40	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 2 µg for 25 µg chromatin	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Recombinant fragment - Human	Dilution info 1/50000	Notes -
Species Human	Dilution info 1/2000	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant fragment - Human	Dilution info -	Notes -

Associated Products

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Target data

See full target information SMAD2

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. Promotes TGFB1-mediated transcription of odontoblastic differentiation genes in dental papilla cells (By similarity). Positively regulates PDPK1 kinase activity by stimulating its dissociation from the 14-3-3 protein YWHAQ which acts as a negative regulator. May act as a tumor suppressor in colorectal carcinoma (PubMed:8752209).

Additional Targets

SMAD3

Alternative names

MADH2, MADR2, SMAD2, Mothers against decapentaplegic homolog 2, MAD homolog 2, Mothers against DPP homolog 2, JV18-1, Mad-related protein 2, SMAD family member 2, hMAD-2, SMAD 2, Smad2, hSMAD2

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR19557
Purification technique: Affinity purification Protein A
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
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 batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Smad2 and Smad3 are intracellular proteins tightly involved in the TGF-β signaling pathway. These proteins also known as mothers against decapentaplegic homolog 2 and 3 play an important role in transmitting signals from the cell surface to the nucleus. Smad2 has a molecular weight of approximately 58 kDa while Smad3 is about 52 kDa. They are expressed in various tissues throughout the body including heart skin and immune cells. Upon activation Smad2 and Smad3 undergo phosphorylation a modification that is important for their function as signal transducers.

Biological function summary

The functions of Smad2 and Smad3 are critical in mediating cellular processes such as proliferation differentiation and apoptosis. These proteins often form complexes specifically oligomeric structures with other Smad proteins like co-Smad4 to exert their regulatory roles. The Smad2/3 complexes control the transcription of specific genes in response to TGF-β stimuli influencing cellular responses in a context-dependent manner.

Pathways

Smad2 and Smad3 are central to the TGF-β signaling pathway one significant pathway that affects cellular growth and differentiation. These proteins interact with TGF-β receptors on the cell surface to become phosphorylated forming a complex with Smad4 which then translocates to the nucleus. In the nucleus this complex regulates gene expression. Both Smad2 and Smad3 also engage with other signaling pathways such as the MAPK pathway further influencing cell behavior and cooperating with proteins like ERK.

Associated diseases and disorders

Smad2 and Smad3 have been linked to conditions like cancer and fibrosis. Dysregulation of Smad2/3 signaling often leads to unchecked cell proliferation a hallmark of tumorigenesis. In fibrosis excessive Smad2/3 activity contributes to tissue scarring due to increased extracellular matrix production. For instance their interaction with proteins like α-SMA (alpha-smooth muscle actin) in fibroblasts plays a role in cardiac and pulmonary fibrosis. Understanding their exact contribution provides insights into potential therapeutic targets for treating these diseases.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

10 product images

Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: Lane 1: 2 seconds; Lane 2: 15 seconds.
Human Smad2 fragment recombinant protein contains aa2-270 with His-Tag^®. Human Smad3 fragment recombinant protein contains aa2-227 with His-Tag^® and GST-tag.
All lanes: Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/50000 dilution
Lane 1: Recombinant protein fragment human Smad2 at 0.01 µg
Lane 2: Recombinant protein fragment human Smad3 at 0.01 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 52 kDa
Observed band size: 38 kDa, 60 kDa
Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Blocking/Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/10000 dilution
Lane 1: HeLa (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg
Lane 2: K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 10 µg
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 10 µg
Lane 4: Human fetal liver lysate at 10 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 52 kDa
Observed band size: 58-62 kDa
Exposure time: 3min
Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Blocking/Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/2000 dilution
All lanes: Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 52 kDa
Observed band size: 58-62 kDa
Exposure time: 15s
Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Blocking/Dilution buffer: 5% NFDM/TBST.
Smad2 and Smad3 can be resolved using a lower percentage gel.
All lanes: Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/10000 dilution
All lanes: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 52 kDa
Observed band size: 52 kDa, 58-62 kDa
Exposure time: 30s
Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Smad2 + Smad3 were immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab207447 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab207447 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10000 dilution.
Lane 1: HeLa whole cell lysate 10μg (Input).
Lane 2: ab207447 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab207447 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 5 seconds.
All lanes: Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
ChIP - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Chromatin was prepared from HaCaT (Human keratinocyte cell line) cells treated with 7ng/ml TGF-β for 1h and non-treated according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab207447 (blue), and 20μl of Anti rabit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
The ChIP condition is designed against Smad2 refer to PMID: 18955504.
Immunocytochemistry/ Immunofluorescence - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 + Smad3 with ab207447 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing signal translocation from cytoplasm to nucleus after TGF-beta (10ng/ml, 1h) treatment in HeLa cells. PMID: 9006934. The nuclear counter stain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab207447 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) secondary at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary at 1/1000 dilution.
Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
ab207447 Immunoprecipitating Smad2 + Smad3 in human HeLa whole cell lysate . 2μg of capture antibody in 0.35mg lysate was used. 10μg of cell lysate was incubated with primary antibody at a dilution of 1/1000 and VeriBlot for IP Detection Reagent (HRP) VeriBlot for IP Detection Reagent (HRP) ab131366 was used for detection at a dilution of 1/1000.
Lane 1: HeLa (human cervix adenocarcinoma) whole cell lysate
Lane 2: HeLa (human cervix adenocarcinoma) whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab207447 in HeLa (human cervix adenocarcinoma) whole cell lysate
Blocking buffer: 5% NFDM/TBST
Diluting buffer: 5% NFDM/TBST
Smad2 and Smad3 can be resolved using a lower percentage gel.
All lanes: Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Observed band size: 58-62 kDa
Exposure time: 3min
Flow Cytometry (Intracellular) - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 and Smad3with ab207447 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/500 dilution was used as the secondary antibody.
ChIC/CUT&RUN sequencing - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HaCaT (Human keratinocyte cell line) cells (treated with 7ng/ml TGF-β for 1h) and 5 µg of ab207447 [EPR19557]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.