Overview

  • Product name

    Human SMAD2 knockout HeLa cell lysate
    See all Smad2 kits
  • Product overview


    Cell line information
    Parental cell line: HeLa
    Organism: Homo sapiens

    Gene editing information
    Editing tool: CRISPR/Cas9
    Mutation: 1 bp deletion in exon2 and 1 bp insertion in exon2.

    Knockout validation: Confirmed by Sanger sequencing and WB.

    Reconstitution instructions: To use as a WB control, resuspend in 45 µL of Sample buffer (40% (w/v) Glycerol, 4% (w/v) Lithium Dodecyl Sulfate, 4% Ficoll 400, 0.025% Phenol Red, 0.025% Brilliant Blue G250, 2 mM EDTA) and 5 µL of DTT to resuspend @ 2mg/ml. Mix well, then boil the sample for 10 minutes before loading it onto the gel.

    User storage instructions: Upon receiving, lysate can be diluted with 1 x SDS sample buffer & will be stable at -20°C for 12 months. Long term storage at -80°C.

  • Notes

    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

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components 1 kit
    Human SMAD2 knockout HeLa cell lysate (Lyophilized) 1 x 100µg
    Human Wild Type HeLa cell lysate (Lyophilized) 1 x 100µg
  • Research areas

  • 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

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.

Images

  • Allel-1: 1 bp deletion in exon2
  • Allel-2: 1 bp insertion in exon2
  • Lane 1: A549 wildtype cell lysate (20 µg)
    Lane 2: Jurkat A549 knockout cell lysate (20 µg)
    Lane 3: HeLa wildtype cell lysate (20 µg)
    Lane 4: SMAD2 HeLa knockout 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 HeLa wildtype. Loss of signal was observed when knockout sample 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.

  • Lane 1: Jurkat cell lysate (20 µg)
    Lane 2: Jurkat A549 knockout cell lysate (20 µg)
    Lane 3: HeLa wildtype cell lysate (20 µg)
    Lane 4: SMAD2 HeLa knockout 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 HeLa wildtype. Loss of signal was observed when knockout sample 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.

References

ab263833 has not yet been referenced specifically in any publications.

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