RecombinantRabMAb

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

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR19557] to Smad2 + Smad3 - ChIP Grade
  • Suitable for: ICC/IF, IP, Flow Cyt, ChIP, WB
  • Reacts with: Human

Overview

  • Product name

    Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade
    See all Smad2 + Smad3 primary antibodies

  • Description

    Rabbit monoclonal [EPR19557] to Smad2 + Smad3 - ChIP Grade

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, IP, Flow Cyt, ChIP, WBmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Recombinant fragment within Human Smad2 + Smad3 aa 1-300. The exact sequence is proprietary.
    Database link: Q15796

  • Positive control

    • WB: Recombinant protein fragment human Smad2; recombinant protein fragment human Smad3; HeLa, K562, A549 and Jurkat whole cell lysates; human fetal liver lysate. ICC/IF: HeLa cells. Flow Cyt: HeLa cells. IP: HeLa whole cell lysate. ChIP: Chromatin was prepared from HaCaT cells treated with 7ng/ml TGF-ß for 1h.

  • 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
    For more information see here.

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

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    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, as well as customer reviews and Q&As.

Properties

Applications

Our Abpromise guarantee covers the use of ab207447 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
ICC/IF 1/500.
IP 1/40.
Flow Cyt 1/500.
ChIP Use 2 µg for 25 µg of chromatin.
WB 1/2000. Detects a band of approximately 58-62 kDa (predicted molecular weight: 52 kDa).

Target

  • Relevance

    SMAD is a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. They mediate the signal of the transforming growth factor (TGF)-beta, and thus regulate multiple cellular processes, such as cell proliferation, apoptosis, and differentiation.

  • Cellular localization

    Cytoplasm. Nucleus. Note: Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with SMAD4.

  • Database links

    • Alternative names

      • hMAD 2 antibody
      • hMAD 3 antibody
      • hSMAD2 antibody
      • hSMAD3 antibody
      • Mad related protein 2 antibody
      • MADH2 antibody
      • MADH3 antibody
      • MADR2 antibody
      • Mothers against DPP homolog 2 antibody
      • Mothers against DPP homolog 3 antibody
      • Sma and Mad related protein 2 antibody
      • SMA and MAD related protein 3 antibody
      • SMAD 2 antibody
      • SMAD 3 antibody
      • SMAD family member 2 antibody
      • SMAD family member 3 antibody
      • smad2+3 antibody
      • smad2/3 antibody
      see all

    Images

    • Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      All lanes : Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/50000 dilution

      Lane 1 : Recombinant protein fragment human Smad2
      Lane 2 : Recombinant protein fragment human Smad3

      Lysates/proteins at 0.01 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: 38,60 kDa
      why is the actual band size different from the predicted?



      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.

    • Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      All lanes : 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
      Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
      Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
      Lane 4 : Human fetal liver 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: 58-62 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/2000 dilution + Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 52 kDa
      Observed band size: 58-62 kDa why is the actual band size different from the predicted?


      Exposure time: 15 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      Western blot - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447) at 1/10000 dilution + HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 52 kDa
      Observed band size: 52,58-62 kDa why is the actual band size different from the predicted?


      Exposure time: 30 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

      Smad2 and Smad3 can be resolved using a lower percentage gel.

    • Immunocytochemistry/ Immunofluorescence - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      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) (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 (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (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) (ab150120) secondary at 1/1000 dilution.

      -ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary at 1/1000 dilution.

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

      Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 and Smad3 with ab207447 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor® 488) at 1/500 dilution was used as the secondary antibody.

    • Immunoprecipitation - Anti-Smad2 + Smad3 antibody [EPR19557] - ChIP Grade (ab207447)
      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) (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 (ab172730) instead of ab207447 in HeLa whole cell lysate.

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 5 seconds.

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

    • ChIP - 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.

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

    References (6)

    Publishing research using ab207447? Please let us know so that we can cite the reference in this datasheet.

    ab207447 has been referenced in 6 publications.

    • Cibi DM  et al. Neural crest-specific deletion of Rbfox2 in mice leads to craniofacial abnormalities including cleft palate. Elife 8:N/A (2019). PubMed: 31241461
    • Yan Y  et al. Nanosized functional miRNA liposomes and application in the treatment of TNBC by silencing Slug gene. Int J Nanomedicine 14:3645-3667 (2019). PubMed: 31190817
    • Matsumoto S  et al. GREB1 induced by Wnt signaling promotes development of hepatoblastoma by suppressing TGFß signaling. Nat Commun 10:3882 (2019). PubMed: 31462641
    • Chen KJ  et al. Bleomycin-enhanced alternative splicing of fibroblast growth factor receptor 2 induces epithelial to mesenchymal transition in lung fibrosis. Biosci Rep 38:N/A (2018). PubMed: 30049844
    • Xiao Y  et al. TGF-ß/MAPK signaling mediates the effects of bone marrow mesenchymal stem cells on urinary control and interstitial cystitis after urinary bladder transplantation. Am J Transl Res 9:1193-1202 (2017). PubMed: 28386345
    • Cai J  et al. Simultaneous overactivation of Wnt/ß-catenin and TGFß signalling by miR-128-3p confers chemoresistance-associated metastasis in NSCLC. Nat Commun 8:15870 (2017). PubMed: 28627514

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