Recombinant
RabMAb

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

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

     

     

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

    This product is a recombinant rabbit monoclonal antibody.

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

  • 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.

  • 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.

  • 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.

  • 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.

  • 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 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.

  • 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.

  • 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.

  • 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.

References

This product has been referenced in:

  • Cibi DM  et al. Neural crest-specific deletion of Rbfox2 in mice leads to craniofacial abnormalities including cleft palate. Elife 8:N/A (2019). Read more (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). Read more (PubMed: 31190817) »
See all 5 Publications for this product

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