Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Smad2 antibody [EP784Y] - BSA and Azide free (ab157371)

Overview

  • Product name

    Anti-Smad2 antibody [EP784Y] - BSA and Azide free
    See all Smad2 primary antibodies
  • Description

    Rabbit monoclonal [EP784Y] to Smad2 - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, Flow Cyt, IHC-P, IP, WBmore details
  • Species reactivity

    Reacts with: Rat, Human
    Does not react with: Mouse
  • Immunogen

    Synthetic peptide corresponding to Human Smad2 aa 50-150.

  • General notes

    The formulation and the concentration of this product is compatible for metal-conjugation for mass cytometry (CyTOF®).

    ab157371 is a PBS only buffer version of ab40855, containing no BSA or sodium azide, ideal for antibody labeling. Please refer to ab40855 for information on protocols, dilutions, and image data.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

Properties

Applications

Our Abpromise guarantee covers the use of ab157371 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
ICC/IF Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG (Low endotoxin, Azide free), is suitable for use as an isotype control with this antibody.

Flow Cyt Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 55 kDa (predicted molecular weight: 58 kDa).

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 [EP784Y] (ab40855) at 1/1000 dilution

    Lane 1 : Wild-type 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



    Lanes 1 - 3: Merged signal (red and green). Green - ab40855 observed at 58 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab40855 was shown to specifically react with Smad2 in wild-type HeLa cells as signal was lost in Smad2 knockout cells. Wild-type and SMAD2 knockout samples were subjected to SDS-PAGE. Ab40855 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40855).

  • ab40855 (purified) at 1/20 immunoprecipitating EGFR in HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate.

    Lane 1 (input): HeLa whole cell lysate (10µg)

    Lane 2 (+): ab40855 + HeLa whole cell lysate.

    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40855 in HeLa whole cell lysate.

    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40855).

  • ab40855 staining Smad2 in the human cell line HeLa (Human epithelial cell line from cervix adenocarcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

    Isoytype control: Rabbit monoclonal IgG (Black)

    Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40855).

  • ab40855 staining Smad2 in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab195889 was used as a counterstain for primary antibody ab40855 at 1/1000. DAPI was used as a nuclear counterstain and PBS as a negative control.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40855).

  • Immunofluorescence staining of HeLa cells with purified ab40855 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was an Alexa Fluor® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40855).

  • Overlay histogram showing PC3 cells stained with ab40855 (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 (ab40855, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40855).

  • ab40855 at a 1:100 dilution staining Smad2 in human prostate carcinoma tissue.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40855).

  • This IHC data was generated using the same anti-Smad2 antibody clone, EP784Y, in a different buffer formulation (cat# ab40855).

    ab40855 stainingSmad2 in human bladder carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/50. A ImmunoHistoProbe one step HRP Polymer was used as a secondary antibody, ready to use.

    Negative control 1: PBS in place of primary antibody.

References

ab157371 has not yet been referenced specifically in any publications.

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