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    products/cell-lines/human-suz12-knockout-hela-cell-line-ab264983.pdf

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Epigenetics and Nuclear Signaling Chromatin Modifying Enzymes Methylation
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Human SUZ12 knockout HeLa cell line (ab264983)

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Western blot - Human SUZ12 knockout HeLa cell line (ab264983)
  • Western blot - Human SUZ12 knockout HeLa cell line (ab264983)
  • Sanger Sequencing - Human SUZ12 knockout HeLa cell line (ab264983)

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Overview

  • Product name

    Human SUZ12 knockout HeLa cell line
    See all SUZ12 lysates
  • Parental Cell Line

    HeLa
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 23 bp deletion in exon 1
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing
  • Tested applications

    Suitable for: WBmore details
  • Biosafety level

    2
  • General notes

    Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

    Recommended control: Human wild-type HeLa cell line (ab255448). 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.

    We will provide viable cells that proliferate on revival.

Properties

  • Number of cells

    1 x 106 cells/vial, 1 mL
  • 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
    • Epigenetics and Nuclear Signaling
    • Chromatin Remodeling
    • Polycomb Silencing
    • PRC2
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • Developmental Families
    • HOX
    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Methylation
    • Lysine methylation

Target

  • Function

    Polycomb group (PcG) protein. Component of the PRC2/EED-EZH2 complex, which methylates 'Lys-9' (H3K9me) and 'Lys-27' (H3K27me) of histone H3, leading to transcriptional repression of the affected target gene. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases, thereby linking two epigenetic repression systems. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1 and CDKN2A.
  • Tissue specificity

    Overexpressed in breast and colon cancer.
  • Involvement in disease

    Note=A chromosomal aberration involving SUZ12 may be a cause of endometrial stromal tumors. Translocation t(7;17)(p15;q21) with JAZF1. The translocation generates the JAZF1-SUZ12 oncogene consisting of the N-terminus part of JAZF1 and the C-terminus part of SUZ12. It is frequently found in all cases of endometrial stromal tumors, except in endometrial stromal sarcomas, where it is rarer.
  • Sequence similarities

    Belongs to the VEFS (VRN2-EMF2-FIS2-SU(Z)12) family.
    Contains 1 C2H2-type zinc finger.
  • Developmental stage

    Expressed at low levels in quiescent cells. Expression rises at the G1/S phase transition.
  • Cellular localization

    Nucleus.
  • Target information above from: UniProt accession Q15022 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 SUZ12 knockout HeLa cell lysate (ab257721)
  • Related Products

    • Anti-SUZ12 antibody (ab12073)
    • Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187)
    • Anti-SUZ12 antibody [EPR5234(N)] - BSA and Azide free (ab249844)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab264983 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: 83 kDa.

Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 83 kDa.

Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

Images

  • Western blot - Human SUZ12 knockout HeLa cell line (ab264983)
    Western blot - Human SUZ12 knockout HeLa cell line (ab264983)
    All lanes : Anti-SUZ12 antibody [EPR5234(N)] - ChIP Grade (ab175187) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : SUZ12 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 83 kDa
    Observed band size: 100 kDa why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab175187 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab175187 was shown to react with SUZ12 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264983 (knockout cell lysate ab257721) lane below 100kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and SUZ12 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab175187 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 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 SUZ12 knockout HeLa cell line (ab264983)
    Western blot - Human SUZ12 knockout HeLa cell line (ab264983)
    All lanes : Anti-SUZ12 antibody (ab12073)

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : SUZ12 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 83 kDa
    Observed band size: 95 kDa why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab12073 observed at 95 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab12073 was shown to react with SUZ12 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264983 (knockout cell lysate ab257721) lane below 95kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and SUZ12 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab12073 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 µg/ml and a 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 SUZ12 knockout HeLa cell line (ab264983)
    Sanger Sequencing - Human SUZ12 knockout HeLa cell line (ab264983)
    Homozygous: 23 bp deletion in exon 1.

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 ab264983? Please let us know so that we can cite the reference in this datasheet.

ab264983 has not yet been referenced specifically in any publications.

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