Recombinant
RabMAb

Recombinant Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445)

Overview

  • Product name

    Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade
    See all Smad2 + Smad3 primary antibodies
  • Description

    Rabbit monoclonal [EPR19557-4] to Smad2 + Smad3 - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ICC/IF, Flow Cyt, IP, ChIPmore details
  • Species reactivity

    Reacts with: Mouse, Rat, 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 and recombinant protein fragment human Smad3; HEK-293, HepG2, HeLa, Jurkat and C6 whole cell lysates; human fetal heart and fetal kidney lysates; mouse brain and heart lysates; rat brain and spleen lysates. ICC/IF: HeLa cells. Flow Cyt: HeLa cells. IP: HeLa whole cell lysate. ChIP: Chromatin 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 ab202445 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
WB 1/1000. Detects a band of approximately 58-62 kDa (predicted molecular weight: 52 kDa).
ICC/IF 1/200.
Flow Cyt 1/600.
IP 1/40.
ChIP Use 2 µg for 25 µg of chromatin.

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 [EPR19557-4] - ChIP Grade (ab202445) at 1/1000 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?


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% NFDM/TBST.

    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 [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution

    Lane 1 : 293 (Human epithelial cell line from embryonic kidney) whole cell lysate
    Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
    Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) 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: 58-62 kDa why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1/2: 10 seconds; Lane 3: 3 seconds; Lane 4: 1 seconds.

  • All lanes : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution

    Lane 1 : Human fetal heart lysate
    Lane 2 : Human fetal kidney lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

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



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1: 30 seconds; Lane 2: 10 seconds.

  • Lane 1 : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/1000 dilution
    Lanes 2 & 5 : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/2000 dilution
    Lanes 3-4 : Anti-Smad2 + Smad3 antibody [EPR19557-4] - ChIP Grade (ab202445) at 1/20000 dilution

    Lane 1 : Mouse brain lysate
    Lane 2 : Mouse heart lysate
    Lane 3 : Rat brain lysate
    Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate
    Lane 5 : Rat spleen 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?



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1: 3 minutes; Lane 2: 10 seconds; Lane 3: 30 seconds; Lane 4: 3 seconds; Lane 5: 5 seconds.

     

  • 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 ab202445 at 1/200 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). The results show signal translocation after TGF-beta (10ng/ml, 1h) treatment on HeLa cells.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: ab202445 at 1/200 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody 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 antibody at 1/1000 dilution.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Smad2 + Smad3 with ab202445 at 1/600 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 1/2000 dilution was used as the secondary antibody.

  • Smad2 + Smad3 was immunoprecipitated from 0.35mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab202445 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab202445 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: ab202445 IP in HeLa whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202445 in HeLa whole cell lysate.

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

    Exposure time: 3 seconds.

  • 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 ab202445 (blue), and 20µl of Anti rabbit 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).

References

This product has been referenced in:

  • Abudureheman A  et al. High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma. J Cancer Res Clin Oncol 144:1025-1035 (2018). Read more (PubMed: 29532228) »
  • Shan L  et al. Epigallocatechin gallate improves airway inflammation through TGF-ß1 signaling pathway in asthmatic mice. Mol Med Rep 18:2088-2096 (2018). Read more (PubMed: 29916550) »
See all 4 Publications for this product

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