Human CDK6 knockout HeLa cell line (ab266059)

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Western blot - Human CDK6 knockout HeLa cell line (ab266059)
  • Western blot - Human CDK6 knockout HeLa cell line (ab266059)
  • Western blot - Human CDK6 knockout HeLa cell line (ab266059)
  • Sanger Sequencing - Human CDK6 knockout HeLa cell line (ab266059)
  • Sanger Sequencing - Human CDK6 knockout HeLa cell line (ab266059)
  • Cell Culture - Human CDK6 knockout HeLa cell line (ab266059)

Overview

  • Product name

    Human CDK6 knockout HeLa cell line
    See all Cdk6 lysates

  • Parental Cell Line

    HeLa

  • Organism

    Human

  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and Insertion of the selection cassette in exon 2

  • 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 -80ºC. Storage at -80ºC may result in loss of viability.

    1. Thaw the vial in 37ºC water bath 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 (Click here to view haemocytometer protocol) or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. This should allow for confluency within 48 hours. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
    4. Incubate the culture at 37ºC 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 for confluency (80-90% confluence) within 48 hours.
    • 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.

    Click here to view the Mammalian cell tissue culture protocol

    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.

Properties

Target

  • Function

    Probably involved in the control of the cell cycle. Interacts with D-type G1 cyclins.

  • Sequence similarities

    Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily.
    Contains 1 protein kinase domain.
  • Information by UniProt

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab266059 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: 37 kDa.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 37 kDa.

Images

  • Western blot - Human CDK6 knockout HeLa cell line (ab266059)
    Western blot - Human CDK6 knockout HeLa cell line (ab266059)
    All lanes : Anti-Cdk6 antibody [98D] (ab241554) at 1/2000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : CDK6 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 37 kDa
    Observed band size: 38 kDa why is the actual band size different from the predicted?



    Lanes 1 - 2: Merged signal (red and green). Green - ab241554 observed at 38 kDa. Red - loading control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55 kDa.

    ab241554 was shown to react with Cdk6 in HeLa wild-type cells in Western blot with loss of signal observed in CDK6 knockout cell line ab266059 (CDK6 knockout cell lysate ab257088). Wild-type HeLa and CDK6 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab241554 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  • Western blot - Human CDK6 knockout HeLa cell line (ab266059)
    Western blot - Human CDK6 knockout HeLa cell line (ab266059)
    All lanes : Anti-Cdk6 antibody (ab54576)

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : CDK6 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 37 kDa
    Observed band size: 37 kDa



    Lanes 1- 2: Merged signal (red and green). Green - ab54576 observed at 37 kDa. Red - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) observed at 50 kDa.

     ab54576 was shown to react with Cdk6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab266059 (knockout cell lysate ab257088) was used. Wild-type HeLa and Cdk6 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. ab54576 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Western blot - Human CDK6 knockout HeLa cell line (ab266059)
    Western blot - Human CDK6 knockout HeLa cell line (ab266059)
    All lanes : Anti-Cdk6 antibody [EPR4515] (ab124821) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : CDK6 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 37 kDa
    Observed band size: 37 kDa



    Lanes 1- 2: Merged signal (red and green). Green - ab124821 observed at 37 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

     ab124821 was shown to react with Cdk6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab266059 (knockout cell lysate ab257088) was used. Wild-type HeLa and Cdk6 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. ab124821 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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.

  • Sanger Sequencing - Human CDK6 knockout HeLa cell line (ab266059)
    Sanger Sequencing - Human CDK6 knockout HeLa cell line (ab266059)

    Allele-1: 1 bp insertion in exon2

     

  • Sanger Sequencing - Human CDK6 knockout HeLa cell line (ab266059)
    Sanger Sequencing - Human CDK6 knockout HeLa cell line (ab266059)

    Allele-2: Insertion of the selection cassette in exon 2.

     

  • Cell Culture - Human CDK6 knockout HeLa cell line (ab266059)
    Cell Culture - Human CDK6 knockout HeLa cell line (ab266059)

    Representative images of CDK6 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.

     

     

References (0)

Publishing research using ab266059? Please let us know so that we can cite the reference in this datasheet.

ab266059 has not yet been referenced specifically in any publications.

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