Recombinant
RabMAb

Recombinant Anti-SMAD5 (phospho S463 + S465) antibody [MMC-1-104-3] (ab92698)

Overview

  • Product name

    Anti-SMAD5 (phospho S463 + S465) antibody [MMC-1-104-3]
    See all SMAD5 primary antibodies
  • Description

    Rabbit monoclonal [MMC-1-104-3] to SMAD5 (phospho S463 + S465)
  • Host species

    Rabbit
  • Specificity

    This antibody may cross-react with Smad1 Phospho (pS463/465) and Smad9 Phospho (pS465/467).
  • Tested applications

    Suitable for: Dot blot, WB, IHC-Pmore details
    Unsuitable for: Flow Cyt or ICC/IF
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human SMAD5 (phospho S463).

  • Positive control

    • WB: HeLa cell lysate treated with BMP-4. IHC-P: Human breast carcinoma, colonic carcinoma, tonsil, hepatocellular carcinoma, cervical carcinoma and glioma tissues.
  • 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 ab92698 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
Dot blot 1/1000.
WB 1/1000 - 1/10000. Predicted molecular weight: 52 kDa.
IHC-P 1/100 - 1/800. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

  • Application notes
    Is unsuitable for Flow Cyt or ICC/IF.
  • Target

    • Function

      Transcriptional modulator activated by BMP (bone morphogenetic proteins) type 1 receptor kinase. SMAD5 is a receptor-regulated SMAD (R-SMAD).
    • Tissue specificity

      Ubiquitous.
    • 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 serine by BMP (bone morphogenetic proteins) type 1 receptor kinase.
      Ubiquitin-mediated proteolysis by SMAD-specific E3 ubiquitin ligase SMURF1.
    • Cellular localization

      Cytoplasm. Nucleus. Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with SMAD4.
    • Information by UniProt
    • Database links

    • Alternative names

      • DKFZp781C1895 antibody
      • DKFZp781O1323 antibody
      • Dwfc antibody
      • hSmad5 antibody
      • JV5 1 antibody
      • JV5-1 antibody
      • MAD homolog 5 antibody
      • MAD, mothers against decapentaplegic homolog 5 antibody
      • MADH 5 antibody
      • MADH5 antibody
      • Mothers against decapentaplegic homolog 5 antibody
      • mothers against decapentaplegic, drosophila, homolog of, 5 antibody
      • Mothers against DPP homolog 5 antibody
      • MusMLP antibody
      • SMA and MAD related protein 5 antibody
      • SMAD 5 antibody
      • SMAD family member 5 antibody
      • SMAD, mothers against DPP homolog 5 antibody
      • Smad5 antibody
      • SMAD5_HUMAN antibody
      see all

    Images

    • All lanes : Anti-SMAD5 (phospho S463 + S465) antibody [MMC-1-104-3] (ab92698) at 1/5000 dilution (purified)

      Lane 1 : Mouse brain tissue lysate
      Lane 2 : Rat brain tissue lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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



      Blocking and dilution buffer: 5% NFDM /TBST.

    • All lanes : Anti-SMAD5 (phospho S463 + S465) antibody [MMC-1-104-3] (ab92698) at 1/10000 dilution (purified)

      Lane 1 : HeLa whole cell lysate - untreated
      Lane 2 : HeLa whole cell lysate - treated with Bone Morphogenetic protein-4

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

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



      Blocking and dilution buffer: 5% NFDM /TBST.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue labelling SMAD5 (phospho S463 + S465) with purified ab92698 at a dilution of 1/800. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling SMAD5 (phospho S463 + S465) with purified ab92698 at a dilution of 1/800. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling SMAD5 (phospho S463 + S465) with purified ab92698 at a dilution of 1/800. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Dot blot analysis of SMAD5 (pS463 + pS465) peptide (Lane 1), SMAD5 (pS465) peptide (Lane 2), SMAD5 (pS463) peptide (Lane 3) and SMAD5 non-phospho peptide (Lane 4) labelling SMAD5 (pS465) with purified ab92698 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

      Blocking and dilution buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

    • All lanes : Anti-SMAD5 (phospho S463 + S465) antibody [MMC-1-104-3] (ab92698) at 1/10000 dilution

      Lane 1 : Untreated HeLa (human cervix adenocarcinoma) whole cell lysates 20µg
      Lane 2 : HeLa (human cervix adenocarcinoma) treated with Bone Morphogenetic Protein-4 (BMP-4) whole cell lysates 20µg
      Lane 3 : HeLa (human cervix adenocarcinoma) treated with Bone Morphogenetic Protein-4 (BMP-4) and phosphatase, whole cell lysates 20µg.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

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


      Exposure time: 30 seconds


      Blocking and dilution buffer: 5% NFDM/TBST.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of (1) human breast carcinoma and (2) human colonic carcinoma tissues labelling SMAD5 (phospho S463 + P465) with unpurified ab92698 at a dilution of 1/100.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human tonsil tissue labelling SMAD5 (phospho S463 + S465) with unpurified ab92698.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human hepatocellular carcinoma tissue labelling SMAD5 (phospho S463 + S465) with unpurified ab92698.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labelling SMAD5 (phospho S463 + S465) with unpurified ab92698.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue labelling SMAD5 (phospho S463 + S465) with unpurified ab92698.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    References

    This product has been referenced in:

    • Frýdlová J  et al. Effect of stimulated erythropoiesis on liver SMAD signaling pathway in iron-overloaded and iron-deficient mice. PLoS One 14:e0215028 (2019). Read more (PubMed: 30958854) »
    • Deng Z  et al. Characterization of articular cartilage homeostasis and the mechanism of superior cartilage regeneration of MRL/MpJ mice. FASEB J N/A:fj201802132RR (2019). Read more (PubMed: 31042406) »
    See all 18 Publications for this product

    Customer reviews and Q&As

    1-3 of 3 Abreviews or Q&A

    Application
    Western blot
    Loading amount
    150 cells
    Gel Running Conditions
    Reduced Denaturing
    Sample
    Ciona intestinalis Cell lysate - whole cell (embryos)
    Specification
    embryos
    Treatment
    100 μM human BMP 4
    Blocking step
    PVDF Blocking Reagent for Can Get Signal (TOYOBO) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 28°C

    Abcam user community

    Verified customer

    Submitted Aug 20 2013

    Answer

    Thank you for your inquiry.



    The sequence of the antigen peptide of this protein is “H2N-KKKNPIS(pS)V(pS)-OH”, Swiss-Prot Q99717.



    Please find the attached file which shows the homology between SMAD5 peptide used for anti-Smad5(phospho S463 + S465) antibody [MMC-1-104-3] between SMAD1 was 70%. Comparing between SMAD5 peptide used for anti-Smad5(phospho S463 + S465) antibody [MMC-1-104-3] and SMAD1, they exactly match except the first three lysines on the N-terminus. Therefore, we suspect this antibody may cross-react with Smad1 Phospho (pS463/465), but we do not have any data to show this nor be able to guarantee it to work.



    If you have any other questions please do not hesitate to contact me.



    Thank you.

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