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Epigenetics and Nuclear Signaling Chromatin Modifying Enzymes Methylation
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Human EHMT2 (G9A) knockout HeLa cell line (ab265149)

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Western blot - Human EHMT2 knockout HeLa cell line (ab265149)
  • Western blot - Human EHMT2 knockout HeLa cell line (ab265149)
  • Sanger Sequencing - Human EHMT2 knockout HeLa cell line (ab265149)
  • Sanger Sequencing - Human EHMT2 knockout HeLa cell line (ab265149)
  • Cell Culture - Human EHMT2 (G9A) knockout HeLa cell line (ab265149)

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Anti-EHMT2/G9A antibody [EPR4019(2)] (ab133482)
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Recombinant human EHMT2/G9A protein (ab167966)
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Anti-EHMT2/G9A antibody [EPR18894] (ab185050)

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Overview

  • Product name

    Human EHMT2 (G9A) knockout HeLa cell line
    See all EHMT2/G9A lysates
  • 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 knockout 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, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

    Culture medium: DMEM (High Glucose) + 10% FBS

    Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.

    1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
    2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
    3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
    4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.

    Subculture guidelines:

    • All seeding densities should be based on cell counts gained by established methods.
    • A guide seeding density of 2x104 cells/cm2 is recommended.
    • A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
    • Cells should be passaged when they have achieved 80-90% confluence.

    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

  • Number of cells

    1 x 106 cells/vial, 1 mL
  • Viability

    ~80%
  • Adherent /Suspension

    Adherent
  • Tissue

    Cervix
  • Cell type

    epithelial
  • Disease

    Adenocarcinoma
  • Gender

    Female
  • STR Analysis

    Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10
  • Antibiotic resistance

    Puromycin 1.00µg/ml
  • Mycoplasma free

    Yes
  • Storage instructions

    Shipped on Dry Ice. Store in liquid nitrogen.
  • Storage buffer

    Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Methylation
    • Stem Cells
    • Embryonic Stem Cells
    • Intracellular
    • Stem Cells
    • Germline Stem Cells
    • Embryonic Germ Cells
    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Methylation
    • Lysine methylation
    • Developmental Biology
    • Reproduction
    • Germ cell markers
    • Developmental Biology
    • Embryogenesis
    • Embryonic stem cells
    • Intracellular

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.
  • Target information above from: UniProt accession Q96KQ7 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt

Associated products

  • KO cell lysates

    • Human EHMT2 (G9A) knockout HeLa cell lysate (ab257080)
  • Related Products

    • Anti-EHMT2/G9A antibody [EPR4019(2)] (ab133482)
    • Anti-EHMT2/G9A antibody [EPR18894] (ab185050)
    • Anti-EHMT2/G9A antibody [EPR18894] - BSA and Azide free (ab240289)
    • Anti-EHMT2/G9A antibody [EPR4019(2)] - BSA and Azide free (ab248517)

Applications

The Abpromise guarantee

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.
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)

    Allele-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)

    Allele-2: 370 bp deletion in exon 7.

     

  • Cell Culture - Human EHMT2 (G9A) knockout HeLa cell line (ab265149)
    Cell Culture - Human EHMT2 (G9A) knockout HeLa cell line (ab265149)

    Representative images of EHMT2 knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, 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

  • Hemocytometer protocol
  • Mammalian cell tissue culture techniques protocol

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

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.

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