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    products/primary-antibodies/smad2-antibody-ep567y-ab33875.pdf

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Signal Transduction Signaling Pathway Nuclear Signaling SMADs
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Smad2 antibody [EP567Y] (ab33875)

  • Datasheet
  • SDS
Reviews (2)Q&A (1)References (42)

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Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Immunocytochemistry/ Immunofluorescence - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] (ab33875)
  • Anti-Smad2 antibody [EP567Y] (ab33875)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP567Y] to Smad2
  • Suitable for: Flow Cyt (Intra), WB, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Human

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Alexa Fluor® 488 Alexa Fluor® 647 Carrier Free

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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: Flow Cyt (Intra), WB, ICC/IFmore details
    Unsuitable for: IHC-P or IP
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Immunogen

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

  • Positive control

    • WB: HeLa, A549, RAW264.7, and Jurkat cell lysate ICC/IF: HeLa cells Flow Cyt (intra): PC3 and Jurkat 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.

    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.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP567Y
  • Isotype

    IgG
  • Research areas

    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • SMADs
    • Epigenetics and Nuclear Signaling
    • Nuclear Signaling Pathways
    • SMADs
    • Stem Cells
    • Signaling Pathways
    • TGF beta
    • Cytoplasmic
    • Cancer
    • Growth factors
    • TGF

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-Smad2 antibody [EP567Y] (ab196320)
    • Alexa Fluor® 647 Anti-Smad2 antibody [EP567Y] (ab196538)
    • Anti-Smad2 antibody [EP567Y] - BSA and Azide free (ab216454)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Corresponding Antibody

    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human SMAD2 knockout HeLa cell line (ab255430)
  • KO cell lysates

    • Human SMAD2 knockout HeLa cell lysate (ab263833)
  • Positive Controls

    • Jurkat whole cell lysate (ab7899)
  • Recombinant Protein

    • Recombinant Human Smad2 protein (ab63176)

Applications

The Abpromise guarantee

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
Flow Cyt (Intra)
1/110.

For unpurified, use 1/70.  ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB (2)
1/1000 - 1/2000. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa).

For unpurified, use 1/1000.

ICC/IF
1/300.
Notes
Flow Cyt (Intra)
1/110.

For unpurified, use 1/70.  ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/1000 - 1/2000. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa).

For unpurified, use 1/1000.

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.
  • Target information above from: UniProt accession Q15796 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 4087 Human
    • Entrez Gene: 17126 Mouse
    • Entrez Gene: 29357 Rat
    • Omim: 601366 Human
    • SwissProt: Q15796 Human
    • SwissProt: Q62432 Mouse
    • SwissProt: O70436 Rat
    • Unigene: 12253 Human
    • Unigene: 705764 Human
    • Unigene: 391091 Mouse
    • Unigene: 2755 Rat
    see all
  • 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

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

    Lane 1 : Wild-type A549 cell lysate
    Lane 2 : Jurkat cell lysate
    Lane 3 : Wild-type HeLa cell lysate
    Lane 4 : SMAD2 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 58 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab33875 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.

     ab33875 was shown to react with Smad2 in wild-type HeLa. Loss of signal was observed when knockout cell line ab255430 (knockout cell lysate ab263833) was used. Wild-type and Smad2 knockout samples were subjected to SDS-PAGE. ab33875 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

     

  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    All lanes : Anti-Smad2 antibody [EP567Y] (ab33875) 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
    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-Smad2 antibody [EP567Y] (ab33875)
    Immunocytochemistry/ Immunofluorescence - Anti-Smad2 antibody [EP567Y] (ab33875)

    Immunocytochemistry/Immunofluorescent analysis of HeLa (human cervix adenocarcinoma epithelial) cells labeling Smad2 with ab33875 at a dilution of 1/500. ab150077, an Alexa Fluor® 488 goat anti-rabbit was used at 1/1000 was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Counterstain antibody: ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200.

    Secondary antibody only negative control is shown in the bottom panels.

    Confocal image showing mainly nuclear staining on HeLa cells after the treatment with TGF-b (10ng/mL) for 1 hour.

  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    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

  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    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

  • Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] (ab33875)
    Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] (ab33875)

    Overlay histogram showingJurkat 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 theisotype control (green line). 

     

     

  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    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

  • Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    Western blot - Anti-Smad2 antibody [EP567Y] (ab33875)
    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

  • Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] (ab33875)
    Flow Cytometry (Intracellular) - Anti-Smad2 antibody [EP567Y] (ab33875)

    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. 

     

     

  • Anti-Smad2 antibody [EP567Y] (ab33875)
    Anti-Smad2 antibody [EP567Y] (ab33875)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (42)

Publishing research using ab33875? Please let us know so that we can cite the reference in this datasheet.

ab33875 has been referenced in 42 publications.

  • Ren C  et al. SHCBP1 Promotes the Progression of Esophageal Squamous Cell Carcinoma Via the TGFß Pathway. Appl Immunohistochem Mol Morphol 29:136-143 (2021). PubMed: 32769441
  • Sun M  et al. MicroRNA-302b mitigates renal fibrosis via inhibiting TGF-ß/Smad pathway activation. Braz J Med Biol Res 54:e9206 (2021). PubMed: 33503202
  • Zhou WJ  et al. NEAT1/miR-200b-3p/SMAD2 axis promotes progression of melanoma. Aging (Albany NY) 12:22759-22775 (2020). PubMed: 33202380
  • Liu D  et al. PRDM16 Upregulation Induced by MicroRNA-448 Inhibition Alleviates Atherosclerosis via the TGF-ß Signaling Pathway Inactivation. Front Physiol 11:846 (2020). PubMed: 32848826
  • Shen W  et al. miR-145-5p attenuates hypertrophic scar via reducing Smad2/Smad3 expression. Biochem Biophys Res Commun 521:1042-1048 (2020). PubMed: 31732152
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-3 of 3 Abreviews or Q&A

Western blot abreview for Anti-Smad2 antibody [EP567Y]

Average
Abreviews
Abreviews
abreview image
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
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Dr. Serban Georgescu

Verified customer

Submitted Mar 03 2008

Western blot abreview for Anti-Smad2 antibody [EP567Y]

Good
Abreviews
Abreviews
abreview image
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%
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Mrs. Birte Münchow

Verified customer

Submitted Oct 17 2006

Question

Hello, I'm looking for antibodies that would let me distinguish Smad2 and Smad3 in paraffin sections of mouse heart. What would you recommend?

Read More

Abcam community

Verified customer

Asked on Oct 19 2012

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 Scientific Support

Answered on Oct 19 2012

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