Overview

  • Product name
    Anti-Smad2 antibody [EP567Y]
    See all Smad2 primary antibodies
  • Description
    Rabbit monoclonal [EP567Y] to Smad2
  • Host species
    Rabbit
  • Specificity
    This antibody detects a region about 40AA before the MH2 region (not the MH2 region itself).
  • Tested applications
    Suitable for: WB, Flow Cyt, ICC/IFmore details
    Unsuitable for: IHC-P or IP
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Smad2 aa 200-300. The exact sequence is proprietary.

  • Positive control
    • Jurkat cell lysate and human adenocarcinoma of uterus
  • 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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab33875 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 - 1/2000. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa).

For unpurified, use 1/1000.

Flow Cyt 1/110.

For unpurified, use 1/70.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/300.
  • Application notes
    Is unsuitable for IHC-P or IP.
  • 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
      modifications
      Phosphorylated 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
    • Alternative names
      • Drosophila, homolog of, MADR2 antibody
      • hMAD-2 antibody
      • HsMAD2 antibody
      • JV18 antibody
      • JV18-1 antibody
      • JV181 antibody
      • MAD antibody
      • MAD homolog 2 antibody
      • MAD Related Protein 2 antibody
      • Mad-related protein 2 antibody
      • MADH2 antibody
      • MADR2 antibody
      • MGC22139 antibody
      • MGC34440 antibody
      • Mother against DPP homolog 2 antibody
      • Mothers against decapentaplegic homolog 2 antibody
      • Mothers against decapentaplegic, Drosophila, homolog of, 2 antibody
      • Mothers against DPP homolog 2 antibody
      • OTTHUMP00000163489 antibody
      • Sma and Mad related protein 2 antibody
      • Sma- and Mad-related protein 2 MAD antibody
      • SMAD 2 antibody
      • SMAD family member 2 antibody
      • SMAD, mothers against DPP homolog 2 antibody
      • SMAD2 antibody
      • SMAD2_HUMAN antibody
      see all

    Images

    • All lanes : Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution

      Lane 1 : Wild-type WT HeLa whole cell lysate
      Lane 2 : SMAD2 knockout HeLa whole cell lysate
      Lane 3 : A549 whole cell lysate

      Lysates/proteins at 20 µg per lane.

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



      Lanes 1 - 3: Merged signal (red and green). Green - ab33875 observed at 52 kDa. Red - loading control, ab9484, observed at 37 kDa.

      ab33875 was shown to specifically react with Smad2 in wild-type WT HeLa cells as signal was lost in SMAD2 knockout cells. Wild-type and SMAD2 knockout samples were subjected to SDS-PAGE. Ab33875 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • Immunofluorescent staining of A673 cells, fixed with 4% PFA, using purified ab33875 at a dilution of 1/300. An Alexa Fluor® 555 goat anti-rabbit was used at 1/200. The negative control is shown in the bottom right hand panel - for the negative control, purified ab33875 was used at a dilution of 1/200 followed by an Alexa Fluor® 555 goat anti-mouse antibody at a dilution of 1/500.

    • Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution (Purified) + RAW264.7 at 10 µg

      Secondary
      HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 58 kDa
      Observed band size: 58 kDa



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • Anti-Smad2 antibody [EP567Y] (ab33875) at 1/2000 dilution (Purified) + Jurkat cell lysate at 10 µg

      Secondary
      HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 58 kDa
      Observed band size: 58 kDa



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • Overlay histogram showing Jurkat cells stained with purified ab33875 (pink line) at a dilution of 1/110. The cells were fixed with 2% PFA. FITC goat anti-rabbit was used at a dilution of 1/150 and rabbit monoclonal IgG was used as the isotype control (green line).

    • Anti-Smad2 antibody [EP567Y] (ab33875) at 1/500 dilution + RAW264.7 cell lysate at 10 µg

      Secondary
      HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 58 kDa
      Additional bands at: 58 kDa. We are unsure as to the identity of these extra bands.



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • Anti-Smad2 antibody [EP567Y] (ab33875) at 1/1000 dilution (Unpurified) + Jurkat cell lysate at 10 µg

      Secondary
      HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 58 kDa
      Additional bands at: 58 kDa. We are unsure as to the identity of these extra bands.



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • All lanes : Anti-Smad2 antibody [EP567Y] (ab33875) at 1/3000 dilution (Unpurified)

      Lane 1 : HGL-5 cells - no additives
      Lane 2 : Cells + activin A
      Lane 3 : Cells + cyclophilin siRNA Control
      Lane 4 : Cells + activin A + cyclophilin
      Lane 5 : non-targeting, negative control for siRNA
      Lane 6 : Cells + activin A, negative control for siRNA
      Lane 7 : Cells + siRNA Smartpool
      Lane 8 : Cells + activin A + siRNA Smartpool
      Lane 9 : Jurkat cells

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/50000 dilution

      Performed under reducing conditions.

      Predicted band size: 58 kDa
      Observed band size: 58 kDa

      See Abreview

    • Overlay histogram showing PC3 cells stained with unpurified ab33875 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab33875, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in PC3 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

    References

    This product has been referenced in:
    • Wu Y  et al. Autocrine transforming growth factor-ß/activin A-Smad signaling induces hepatic progenitor cells undergoing partial epithelial-mesenchymal transition states. Biochimie 148:87-98 (2018). Read more (PubMed: 29544731) »
    • Cheng Z  et al. The long non-coding RNA uc.4 influences cell differentiation through the TGF-beta signaling pathway. Exp Mol Med 50:e447 (2018). WB . Read more (PubMed: 29504607) »
    See all 19 Publications for this product

    Customer reviews and Q&As

    1-3 of 3 Abreviews or Q&A

    Answer

    Thank you for contacting us.

    We do have a number of anti-SMAD2 and anti-SMAD3 antibodies which we guarantee in IHC-P on mouse. We to don't have the opportunity to test specific products against one another in each application but based on Abreviews, publications and other data available on our datasheets I can recommend the following products.

    For identifying both SMAD2 and SMAD3 in IHC-P https://www.abcam.com/Smad2-Smad3-antibody-ab65847.html https://www.abcam.com/Smad2-Smad3-antibody-ab65847.html is guaranteed by our Abpromise in mouse.

    While we have not tested our SMAD2 products for SMAD3 reactivity or the SMAD3 against SMAD2, I can recommend the following products for identifying the specific proteins based on sequence homologies.

    For identifying only SMAD2 in IHC-P we recommend the rabbit monoclonal https://www.abcam.com/Smad2-antibody-EP567Y-ab33875.html https://www.abcam.com/Smad2-antibody-EP567Y-ab33875.html. The immunogen used for this product has only a 44.4% identity to SMAD3 Mouse.

    For identifying only SMAD2 in IHC-P we recommend the rabbit monoclonal https://www.abcam.com/Smad3-antibody-EP568Y-ab40854.html https://www.abcam.com/Smad3-antibody-EP568Y-ab40854.html. The immunogen used for this product has only a 47.4% identity to SMAD2 Mouse.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (c3h10t1/2)
    Loading amount
    25 µg
    Specification
    c3h10t1/2
    Treatment
    TGFbeta 15 and 30 minutes
    Gel Running Conditions
    Reduced Denaturing (4-12% gradient)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Dr. Serban Georgescu

    Verified customer

    Submitted Mar 03 2008

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Human cell line HGL-5)
    Loading amount
    15 µg
    Specification
    Human cell line HGL-5
    Blocking step
    Milk as blocking agent for 15 hour(s) and 0 minute(s) · Concentration: 5%

    Mrs. Birte Münchow

    Verified customer

    Submitted Oct 17 2006

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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