Overview

  • Product name

    Anti-SMAD1+SMAD5 antibody [AF10B7]
  • Description

    Mouse monoclonal [AF10B7] to SMAD1+SMAD5
  • Host species

    Mouse
  • Tested applications

    Suitable for: ChIP, Flow Cyt, WB, ELISAmore details
  • Species reactivity

    Reacts with: Rat, Human
  • Immunogen

    Recombinant human protein purified from E.coli (His-Smad1)

  • Positive control

    • SK-N-MC, Ramos, 293T and PC12 cell lysates.
  • General notes

    This product was changed from ascites to tissue culture supernatant on 18th September 2017. Lot numbers higher than GR188031 will be from tissue culture supernatant. Please note that the dilutions may need to be adjusted accordingly.

     

Properties

Applications

Our Abpromise guarantee covers the use of ab75273 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
ChIP Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

 

WB Use a concentration of 1 µg/ml. Predicted molecular weight: 52 kDa.
ELISA Use at an assay dependent concentration.

Target

  • Relevance

    The Smad family of proteins function in the transmission of extracellular signals in the TGF-ß signaling pathway. Binding of TGF-ß superfamily ligands to extracellular receptors triggers phosphorylation of Smad2 at a Serine-Serine-Methionine-Serine (SSMS) motif at its C-terminus. Phosphorylated Smad2 is then able to form a complex with Smad4. These complexes accumulate in the cell nucleus, where they directly participate in the regulation of gene expression. In mammals, eight Smad proteins have been identified to date. The Smad family of proteins can be divided into three functional groups: the receptor-activated Smads (R-Smads), common mediator Smads (Co-Smads), and the inhibitory Smads (I-Smads). The R-Smads are directly phosphorylated by the activated type I receptors on their C-terminal Ser-Ser-X-Ser (SSXS) motif and include Smad1, Smad2, Smad3, Smad5, and Smad8. Smad2 and Smad3 are phosphorylated in response to TGF-ß and activin, whereas Smad1, Smad5, and Smad8 are phosphorylated in response to BMP (Bone Morphogenetic Protein). This C-terminal phosphorylation allows R-Smad binding to Co-Smad, Smad4, and translocation to the nucleus where they regulate TGF-ß target genes. Smad6 and Smad7 belong to the I-Smad family which bind to the type I receptor or Smad4 and block their interaction with R-Smads.
  • Cellular localization

    Cytoplasm. Nucleus. Note=Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with SMAD4.
  • Database links

  • Alternative names

    • MAD mothers against decapentaplegic homolog 1 antibody
    • MAD mothers against decapentaplegic homolog 5 antibody
    • MADH1 antibody
    • MADH5 antibody
    • Mothers against DPP homolog 1 antibody
    • Mothers against DPP homolog 5 antibody
    • SMAD family member 1 antibody
    • SMAD family member 5 antibody
    • SMAD1 antibody
    • SMAD5 antibody
    see all

Images

  • All lanes : Anti-SMAD1+SMAD5 antibody [AF10B7] (ab75273) at 1/1000 dilution

    Lane 1 : SK-N-MC cell lysate
    Lane 2 : Ramos cell lysate
    Lane 3 : 293T cell lysate
    Lane 4 : PC12 cell lysate

    Predicted band size: 52 kDa
    Observed band size: 58 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 120 kDa. We are unsure as to the identity of these extra bands.

  • Overlay histogram showing HeLa cells stained with ab75273 (red line). The cells were fixed with 80% 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 (ab75273, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

References

This product has been referenced in:

  • Wen Q  et al. 8-bromo-7-methoxychrysin suppress stemness of SMMC-7721 cells induced by co-culture of liver cancer stem-like cells with hepatic stellate cells. BMC Cancer 19:224 (2019). Read more (PubMed: 30866863) »
  • Zhang Y  et al. Selective compounds enhance osteoblastic activity by targeting HECT domain of ubiquitin ligase Smurf1. Oncotarget 8:50521-50533 (2017). Read more (PubMed: 28881580) »
See all 9 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Primary rib chondrocytes)
Gel Running Conditions
Reduced Denaturing (10% acrylamide gel)
Loading amount
20 µg
Treatment
50 ng/mL BMP-2 for 1 hour
Specification
Primary rib chondrocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Jul 12 2016

Answer

Thank you for contacting us.

Ab75273 can detect smad1 and smad5. It’s validated with each recombinant protein.

The molecular weight of smad1 and smad5 is same and that’s the reason why the predicted band size is same.

The possibility of detecting sharing region of smad1 and smad5 is very high because their sequence alignment is 89%.

However, we cannot officially comment which region it detects because it’s not mapped the epitope this antibody detect.

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

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Question
Answer

Thank you for contacting us. Of the three antibodies we discussed yesterday, ab80255 is the best choice, given that we know the immunogen is derived from the N terminus, and your detection antibody is directed against the C terminus. The epitope that ab75273 binds to is unmapped, and so I am concerned that it may not be compatible in a sandwich ELISA with your detection antibody if, by chance, it binds to the C terminus. Antibody ab72504 was actually a duplicate entry of ab80255 and has been removed from the catalogue. Ab80255 has not, however, been tested in ELISA to capture native SMAD1. As I expected, it was validated for ELISA against the peptide that was used as the immunogen. It has been validated for IHC, though, so we expect it will be capable of binding the N terminus when SMAD1 is in its native conformation. Another antibody to consider is ab63356. The situation is the same as for ab80255: untested in ELISA against native, endogenous SMAD1, only against the peptide, but it gives a signal in IHC and ICC. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

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