Human EHMT2 (G9A) knockout HeLa cell line (ab265149)

Submit a question

Overview

  • Product name

    Human EHMT2 (G9A) knockout HeLa cell line

  • Parental Cell Line

    HeLa

  • Organism

    Human

  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 2 bp deletion in exon 7 and 370 bp deletion in exon 7

  • Passage number

    <20

  • Knockout validation

    Sanger Sequencing, Western Blot (WB)

  • Tested applications

    Suitable for: WBmore details

  • Biosafety level

    2

  • General notes

    Recommended control: Human Wild Type HeLa cell line (ab255928)

    Please note a wild type cell line is not automatically included with a KO cell line order, if required please add recommended wild type cell line at no additional cost using the code WILDTYPE-TMTK1

    Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, supplemented with 10% (v/v) DMSO

    Handling procedure: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C will result in loss of viability.
    1. Thaw the vial in 37°C water bath approximately 1-2 minutes.
    2. Transfer the cell suspension to a 15 mL conical tube with pre-warmed 5 mL complete medium DMEM+10% FBS, spin 125×g for approximately 5 minutes at room temperature.
    3. Resuspend the cell pellet with 1 mL pre-warmed complete medium DMEM+10% FBS and dispense into a 25 cm2 culture flask containing 10 mL pre-warmed complete complete medium DMEM+10% FBS.
    4. Incubate the culture at 37°C incubator with 5% CO2.
    5. A subcultivation ratio of 1:4-1:6 is recommended. Cells should be passaged when cells grow splitting at 80-90% confluence.

    Click here to view the Mammalian cell tissue culture protocol

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

Properties

Target

  • Function

    Histone methyltransferase that specifically mono- and dimethylates 'Lys-9' of histone H3 (H3K9me1 and H3K9me2, respectively) in euchromatin. H3K9me represents a specific tag for epigenetic transcriptional repression by recruiting HP1 proteins to methylated histones. Also mediates monomethylation of 'Lys-56' of histone H3 (H3K56me1) in G1 phase, leading to promote interaction between histone H3 and PCNA and regulating DNA replication. Also weakly methylates 'Lys-27' of histone H3 (H3K27me). Also required for DNA methylation, the histone methyltransferase activity is not required for DNA methylation, suggesting that these 2 activities function independently. Probably targeted to histone H3 by different DNA-binding proteins like E2F6, MGA, MAX and/or DP1. May also methylate histone H1. In addition to the histone methyltransferase activity, also methylates non-histone proteins: mediates dimethylation of 'Lys-373' of p53/TP53. Also methylates CDYL, WIZ, ACIN1, DNMT1, HDAC1, ERCC6, KLF12 and itself.

  • Tissue specificity

    Expressed in all tissues examined, with high levels in fetal liver, thymus, lymph node, spleen and peripheral blood leukocytes and lower level in bone marrow.

  • Sequence similarities

    Belongs to the class V-like SAM-binding methyltransferase superfamily. Histone-lysine methyltransferase family. Suvar3-9 subfamily.
    Contains 7 ANK repeats.
    Contains 1 post-SET domain.
    Contains 1 pre-SET domain.
    Contains 1 SET domain.

  • Domain

    The SET domain mediates interaction with WIZ.
    The ANK repeats bind H3K9me1 and H3K9me2.

  • Post-translational
    modifications

    Methylated at Lys-185; automethylated.

  • Cellular localization

    Nucleus. Chromosome. Associates with euchromatic regions. Does not associate with heterochromatin.
  • Information by UniProt

Applications

Our Abpromise guarantee covers the use of ab265149 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. Predicted molecular weight: 132 kDa.

Images

  • Western blot - Human EHMT2 knockout HeLa cell line (ab265149)
    Western blot - Human EHMT2 knockout HeLa cell line (ab265149)
    All lanes : Anti-EHMT2/G9A antibody [EPR18894] (ab185050) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : EHMT2 knockout HeLa cell lysate
    Lane 3 : HEK-293 cell lysate
    Lane 4 : HepG2 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution

    Predicted band size: 132 kDa
    Observed band size: 160 kDa
    why is the actual band size different from the predicted?



    Lanes 1-4: Merged signal (red and green). Green - ab185050 observed at 160 kDa. Red - loading control ab8245 observed at 37 kDa.

     ab185050 Anti-EHMT2/G9A antibody [EPR18894] was shown to specifically react with EHMT2/G9A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265149 (knockout cell lysate ab257080) was used. Wild-type and EHMT2/G9A knockout samples were subjected to SDS-PAGE. ab185050 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 EHMT2 knockout HeLa cell line (ab265149)
    Western blot - Human EHMT2 knockout HeLa cell line (ab265149)
    All lanes : Anti-EHMT2/G9A antibody [EPR4019(2)] (ab133482) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : EHMT2 knockout HeLa cell lysate
    Lane 3 : HEK-293 cell lysate
    Lane 4 : HepG2 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution

    Predicted band size: 132 kDa
    Observed band size: 160 kDa why is the actual band size different from the predicted?



    Lanes 1-4: Merged signal (red and green). Green - ab133482 observed at 160 kDa. Red - loading control ab8245 observed at 37 kDa.

     ab133482 Anti-EHMT2/G9A antibody [EPR4019(2)] was shown to specifically react with EHMT2/G9A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265149 (knockout cell lysate ab257080) was used. Wild-type and EHMT2/G9A knockout samples were subjected to SDS-PAGE. ab133482 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.

     

  • Sanger Sequencing - Human EHMT2 knockout HeLa cell line (ab265149)
    Sanger Sequencing - Human EHMT2 knockout HeLa cell line (ab265149)
    Allel-1: 2 bp deletion in exon 7.
  • Sanger Sequencing - Human EHMT2 knockout HeLa cell line (ab265149)
    Sanger Sequencing - Human EHMT2 knockout HeLa cell line (ab265149)
    Allel-2: 370 bp deletion in exon 7.
  • Other - Human EHMT2 knockout HeLa cell line (ab265149)
    Other - Human EHMT2 knockout HeLa cell line (ab265149)
    Representative images of HeLa knockout, low and high confluency (top left and right respectively) and HeLa wild-type, low and high confluency (bottom left and right respectively), showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.

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

Publishing research using ab265149? Please let us know so that we can cite the reference in this datasheet.

ab265149 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Q&A
Submit a question

There are currently no Questions for ab265149.
Please use the link above to contact us or submit feedback about this product.

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com