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Explore the power of knock-out cell lines for your research

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    human-podxl-knockout-hct116-cell-line-ab266887.pdf

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Signal Transduction Cytoskeleton / ECM Extracellular Matrix Structures Focal Adhesions
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Human PODXL knockout HCT116 cell line (ab266887)

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Western blot - Human PODXL knockout HCT116 cell line (ab266887)
  • Sanger Sequencing - Human PODXL knockout HCT116 cell line (ab266887)
  • Cell Culture - Human PODXL knockout HCT116 cell line (ab266887)

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Overview

  • Product name

    Human PODXL knockout HCT116 cell line
    See all PODXL lysates
  • Parental Cell Line

    HCT116
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 2
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing, Western Blot (WB)
  • Tested applications

    Suitable for: WBmore details
  • Biosafety level

    1
  • General notes

    Recommended control: Human wild-type HCT116 cell line (ab255451). 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: McCoY5a + 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, 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

  • Number of cells

    1 x 106 cells/vial, 1 mL
  • Viability

    ~80%
  • Adherent /Suspension

    Adherent
  • Tissue

    Colon
  • Cell type

    epithelial
  • Disease

    Carcinoma
  • Gender

    Male
  • STR Analysis

    Amelogenin X D5S818: 10, 11 D13S317: 10, 12 D7S820: 11, 12 D16S539: 11, 13 vWA: 17, 22 TH01: 8,9 TPOX: 8, 9 CSF1PO: 7, 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

    • Signal Transduction
    • Cytoskeleton / ECM
    • Extracellular Matrix
    • Structures
    • Focal Adhesions
    • Stem Cells
    • Hematopoietic Progenitors
    • Surface Molecules
    • Cancer
    • Tumor biomarkers
    • Other
    • Cardiovascular
    • Vasculature
    • Endothelium
    • Cardiovascular
    • Angiogenesis
    • Endothelial Cell Markers

Target

  • Function

    Involved in the regulation of both adhesion and cell morphology and cancer progression. Function as an anti-adhesive molecule that maintains an open filtration pathway between neighboring foot processes in the podocyte by charge repulsion. Acts as a pro-adhesive molecule, enhancing the adherence of cells to immobilized ligands, increasing the rate of migration and cell-cell contacts in an integrin-dependent manner. Induces the formation of apical actin-dependent microvilli. Involved in the formation of a preapical plasma membrane subdomain to set up inital epithelial polarization and the apical lumen formation during renal tubulogenesis. Plays a role in cancer development and aggressiveness by inducing cell migration and invasion through its interaction with the actin-binding protein EZR. Affects EZR-dependent signaling events, leading to increased activities of the MAPK and PI3K pathways in cancer cells.
  • Tissue specificity

    Glomerular epithelium cell (podocyte).
  • Sequence similarities

    Belongs to the podocalyxin family.
  • Domain

    Both the O-glycan-rich domain of the extracellular domain and the C-terminus PDZ-binding motif (DTHL) in the cytoplasmic tail harbor an apical sorting signal. The cytoplasmic domain is necessary for the apical membrane targeting and renal tubulogenesis. The cytoplasmic C-terminus PDZ-binding motif (DTHL) is essential for interaction with SLC9A3R1 and for targeting SLC9A3R1 to the apical cell membrane. The extracellular domain is necessary for microvillus formation (By similarity). The large highly anionic extracellular domain allows to maintain open filtration pathways between neighboring podocyte foot processes.
  • Post-translational
    modifications

    N- and O-linked glycosylated. Sialoglycoprotein.
  • Cellular localization

    Apical cell membrane. Cell projection, lamellipodium. Cell projection, filopodium. Cell projection, ruffle. Cell projection, microvillus. Membrane raft. Membrane. In single attached epithelial cells is restricted to a preapical pole on the free plasma membrane whereas other apical and basolateral proteins are not yet polarized. Colocalizes with SLC9A3R2 at the apical plasma membrane during epithelial polarization. Colocalizes with SLC9A3R1 at the trans-Golgi network (transiently) and at the apical plasma membrane. Its association with the membrane raft is transient. Colocalizes with actin filaments, EZR and SLC9A3R1 in a punctate pattern at the apical cell surface where microvilli form. Colocalizes with EZR and SLC9A3R2 at the apical cell membrane of glomerular epithelium cells (By similarity). Forms granular, punctuated pattern, forming patches, preferentially adopting a polar distribution, located on the migrating poles of the cell or forming clusters along the terminal ends of filipodia establishing contact with the endothelial cells. Colocalizes with the submembrane actin of lamellipodia, particularly associated with ruffles. Colocalizes with vinculin at protrusions of cells. Colocalizes with ITGB1. Colocalizes with PARD3, PRKCI, EXOC5, OCLN, RAB11A and RAB8A in apical membrane initiation sites (AMIS) during the generation of apical surface and luminogenesis (By similarity).
  • Target information above from: UniProt accession O00592 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Form

    There are 2 isoforms produced by alternative splicing.

Associated products

  • KO cell lysates

    • Human PODXL knockout HCT116 cell lysate (ab257211)
  • Related Products

    • Anti-PODXL antibody [EPR9518] (ab150358)
    • Anti-PODXL antibody [EPR9518] - BSA and Azide free (ab269888)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab266887 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: 58 kDa.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 58 kDa.

Images

  • Western blot - Human PODXL knockout HCT116 cell line (ab266887)
    Western blot - Human PODXL knockout HCT116 cell line (ab266887)
    All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution

    Lane 1 : Wild-type HCT116 cell lysate
    Lane 2 : PODXL knockout HCT116 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 58 kDa
    Observed band size: 200 kDa why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab150358 observed at 200 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

     ab150358 was shown to react with PODXL in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266887 (knockout cell lysate ab257211) was used. Wild-Type HCT116 and PODXL knockout HCT116 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. ab150358 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 PODXL knockout HCT116 cell line (ab266887)
    Sanger Sequencing - Human PODXL knockout HCT116 cell line (ab266887)
    Homozygous: 1 bp insertion in exon2
  • Cell Culture - Human PODXL knockout HCT116 cell line (ab266887)
    Cell Culture - Human PODXL knockout HCT116 cell line (ab266887)
    Representative images PODXL knockout HCT116 cells, low and high confluency examples (top left and right respectively) and wild-type HCT116 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 M5000 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 ab266887? Please let us know so that we can cite the reference in this datasheet.

ab266887 has not yet been referenced specifically in any publications.

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