RecombinantRabMAb

Recombinant Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)

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Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
  • Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
  • Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
  • Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
  • Immunocytochemistry/ Immunofluorescence - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
  • Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP728(2)AY] to SMAD5 (phospho S463 + S465)
  • Suitable for: WB, ICC/IF
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY]
    See all SMAD5 primary antibodies

  • Description

    Rabbit monoclonal [EP728(2)AY] to SMAD5 (phospho S463 + S465)

  • Host species

    Rabbit

  • Specificity

    Detects SMAD5 phosphorylated at Serine 463 and 465. This antibody may cross-react with Smad1 Phospho (pS463/465) and Smad9 Phospho (pS465/467).

    Stimulation may be required to allow detection of the phosphorylated protein.  Please see images below for recommended treatment conditions and positive controls.

  • Tested applications

    Suitable for: WB, ICC/IFmore details
    Unsuitable for: Flow Cyt,IHC-P or IP

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • HeLa cytoplasmic or nuclear lysate treated with BMP-4 HeLa cells

  • 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.

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab76296 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)
1/500. Predicted molecular weight: 52 kDa.
ICC/IF
1/50 - 1/100.
Notes
WB
1/500. Predicted molecular weight: 52 kDa.
ICC/IF
1/50 - 1/100.
Application notes
Is unsuitable for Flow Cyt,IHC-P or IP.

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

    see all

  • 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

  • Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    All lanes : Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296) at 1/1000 dilution

    Lane 1 : Untreated C6 (Rat glial tumor glial cell) whole cell lysate with NFDM/TBST
    Lane 2 : C6 treated with 10ng/ml TGF beta 3 and 50µM MG-132 for 30 min whole cell lysate with NFDM/TBST
    Lane 3 : C6 treated with 10ng/ml TGF beta 3 and 50µM MG-132 for 30 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour with NFDM/TBST

    Lysates/proteins at 15 µg per lane.

    Blocking peptides at 5 % per lane.

    Secondary
    Lane 1 : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution
    Lanes 2-3 : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051)

    Predicted band size: 52 kDa


    Exposure time: 7 seconds
  • Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    All lanes : Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296) at 1/1000 dilution

    Lane 1 : Untreated HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with NFDM/TBST
    Lane 2 : HeLa treated with 20ng/ml TGF beta 1 for 15 min whole cell lysate with NFDM/TBST
    Lane 3 : HeLa treated with 20ng/ml TGF beta 1 for 15 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour with NFDM/TBST

    Lysates/proteins at 15 µg per lane.

    Blocking peptides at 5 % per lane.

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

    Predicted band size: 52 kDa


    Exposure time: 20 seconds
  • Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    All lanes : Anti-SRGAP1 antibody (ab76926) at 1/1000 dilution

    Lane 1 : Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate with NFDM/TBST
    Lane 2 : NIH/3T3 treated with 20ng/ml TGF beta 1 and 50µM MG-132 for 15 min whole cell lysate with NFDM/TBST
    Lane 3 : NIH/3T3 treated with 20ng/ml TGF beta 1 and 50µM MG-132 for 15 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour with NFDM/TBST

    Lysates/proteins at 15 µg per lane.

    Blocking peptides at 5 % per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

    Predicted band size: 52 kDa
    Observed band size: 52 kDa


    Exposure time: 100 seconds
  • Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    Western blot - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    All lanes : Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296) at 1/500 dilution

    Lane 1 : HeLa cell lysate - untreated
    Lane 2 : HeLa cell lysate - treated with BMP-4

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : goat anti-rabbit HRP at 1/2000 dilution

    Predicted band size: 52 kDa
    Observed band size: 52 kDa

  • Immunocytochemistry/ Immunofluorescence - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    Immunocytochemistry/ Immunofluorescence - Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    Immunofluorescent staining of HeLa cells using ab76296 at a 1/50-1/100 dilution.
  • Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)
    Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY] (ab76296)

References (4)

Publishing research using ab76296? Please let us know so that we can cite the reference in this datasheet.

ab76296 has been referenced in 4 publications.

  • Zhou Q  et al. A New Insight of Kartogenin Induced the Mesenchymal Stem Cells (MSCs) Selectively Differentiate into Chondrocytes by Activating the Bone Morphogenetic Protein 7 (BMP-7)/Smad5 Pathway. Med Sci Monit 25:4960-4967 (2019). PubMed: 31271564
  • Cui Y  et al. Single-Cell Transcriptome Analysis Maps the Developmental Track of the Human Heart. Cell Rep 26:1934-1950.e5 (2019). PubMed: 30759401
  • Ghiabi P  et al. Breast cancer cells promote a notch-dependent mesenchymal phenotype in endothelial cells participating to a pro-tumoral niche. J Transl Med 13:27 (2015). WB ; Human . PubMed: 25623554
  • Nili M  et al. Soluble Repulsive Guidance Molecule c/Hemojuvelin Is a Broad Spectrum Bone Morphogenetic Protein (BMP) Antagonist and Inhibits both BMP2- and BMP6-mediated Signaling and Gene Expression. J Biol Chem 285:24783-92 (2010). WB ; Human . PubMed: 20530805

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Western blot abreview for Anti-SMAD5 (phospho S463 + S465) antibody [EP728(2)AY]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Human prostate and cervical cancer cell)
Gel Running Conditions
Reduced Denaturing (7.5%)
Loading amount
30 µg
Specification
Human prostate and cervical cancer cell
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Aug 20 2015

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