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Human SMAD2 knockout HeLa cell lysate (ab263833)

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Western blot - Human SMAD2 knockout HeLa cell lysate
  • Western blot - Human SMAD2 knockout HeLa cell lysate (ab263833)
  • Western blot - Human SMAD2 knockout HeLa cell lysate (ab263833)
  • Sanger Sequencing - Human SMAD2 knockout HeLa cell lysate (ab263833)
  • Sanger Sequencing - Human SMAD2 knockout HeLa cell lysate (ab263833)

Overview

  • Product name

    Human SMAD2 knockout HeLa cell lysate
    See all Smad2 kits

  • Product overview


    Knockout cell lysate achieved by CRISPR/Cas9.

  • Parental Cell Line

    HeLa

  • Organism

    Human

  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon2 and 1 bp insertion in exon2.

  • Passage number

    <20

  • Knockout validation

    Sanger Sequencing, Western Blot (WB)

  • Reconstitution notes

    To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.

    *Usage of SDS sample buffer is not recommended with these lyophilized lysates.

  • Notes

    Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.

    User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.

    Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
    See here for more information on knockout cell lysates.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

    This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

  • Tested applications

    Suitable for: WBmore details

Properties

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

  • Alternative names

    • Drosophila, homolog of, MADR2
    • hMAD-2
    • HsMAD2
    • JV18
    • JV18-1
    • JV181
    • MAD
    • MAD homolog 2
    • MAD Related Protein 2
    • Mad-related protein 2
    • MADH2
    • MADR2
    • MGC22139
    • MGC34440
    • Mother against DPP homolog 2
    • Mothers against decapentaplegic homolog 2
    • Mothers against decapentaplegic, Drosophila, homolog of, 2
    • Mothers against DPP homolog 2
    • OTTHUMP00000163489
    • Sma and Mad related protein 2
    • Sma- and Mad-related protein 2 MAD
    • SMAD 2
    • SMAD family member 2
    • SMAD, mothers against DPP homolog 2
    • SMAD2
    • SMAD2_HUMAN
    see all

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab263833 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
Use at an assay dependent concentration.
Notes
WB
Use at an assay dependent concentration.

Images

  • Western blot - Human SMAD2 knockout HeLa cell lysate
    Western blot - Human SMAD2 knockout HeLa cell lysate

    Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

    Lane 1: Wild-type HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate, 20 ug

    Lane 2: Wild-type HeLa (human cervix adenocarcinoma epithelial cell) treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate, 20 ug

    Lane 3: Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate, 20 ug

    Lane 4: Smad2 knockout HeLa (human cervix adenocarcinoma epithelial cell), treated with 20 ng/ml TGF beta1 for 15 minutes, whole cell lysate, 20 ug

    Lanes 1-4: Merged signal (red and green). Green - ab280888 observed at 60 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

    ab280888 Anti-Smad2 (phospho S467) antibody [EPR23681-40] was shown to specifically react with Smad2 in wild-type HeLa cells. 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. 

    ab280888 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4? overnight 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 10000 dilution for 1 hour at room temperature before imaging.

  • Western blot - Human SMAD2 knockout HeLa cell lysate (ab263833)
    Western blot - Human SMAD2 knockout HeLa cell lysate (ab263833)

    Lane 1: A549 cell lysate (20 µg)

    Lane 2: Jurkat cell lysate (20 µg)

    Lane 3: Wild-type HeLa cell lysate (20 µg)

    Lane 4: SMAD2 knockout HeLa cell lysate (20 µg)

    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 - Human SMAD2 knockout HeLa cell lysate (ab263833)
    Western blot - Human SMAD2 knockout HeLa cell lysate (ab263833)

    Lane 1: A549 cell lysate (20 µg)

    Lane 2: Jurkat cell lysate (20 µg)

    Lane 3: Wild-type HeLa cell lysate (20 µg)

    Lane 4: SMAD2 knockout HeLa cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab40855 observed at 58 kDa. Red - loading control, ab8245 observed at 37 kDa.  

    ab40855 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. ab40855 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 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.

  • Sanger Sequencing - Human SMAD2 knockout HeLa cell lysate (ab263833)
    Sanger Sequencing - Human SMAD2 knockout HeLa cell lysate (ab263833)

    Allele-1: 1 bp deletion in exon2

     

  • Sanger Sequencing - Human SMAD2 knockout HeLa cell lysate (ab263833)
    Sanger Sequencing - Human SMAD2 knockout HeLa cell lysate (ab263833)

    Allele-2: 1 bp insertion in exon2

     

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

References (0)

Publishing research using ab263833? Please let us know so that we can cite the reference in this datasheet.

ab263833 has not yet been referenced specifically in any publications.

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