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Human PDGFRA knockout SH-SY5Y cell line (ab275335)

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Western blot - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
  • Immunocytochemistry - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
  • Western blot - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
  • Sanger Sequencing - Human PDGFRA knockout SH-SY5Y cell line (ab275335)

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Anti-PDGFR alpha antibody [EPR5480] (ab134123)
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Anti-PDGFR alpha antibody [EPR22059-270] (ab203491)

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Overview

  • Product name

    Human PDGFRA knockout SH-SY5Y cell line
    See all PDGFR alpha lysates
  • Parental Cell Line

    SHSY-5Y
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 67 bp deletion in exon 3
  • Passage number

    <20
  • Knockout validation

    Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB)
  • Tested applications

    Suitable for: WB, ICCmore details
  • Biosafety level

    1
  • General notes

    Recommended control: Human wild-type SHSY-5Y cell line (ab275475). 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: 1:1 mixture of EMEM and F-12K + 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 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

    ~90%
  • Tissue

    Bone marrow
  • Cell type

    neuroblastoma
  • Disease

    Neuroblastoma
  • Gender

    Female
  • Mycoplasma free

    Yes
  • Storage instructions

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

    Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
  • Purity

    Immunogen affinity purified
  • Research areas

    • Cardiovascular
    • Angiogenesis
    • Growth Factors
    • Other
    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • Receptor Tyrosine Kinases
    • Signal Transduction
    • Growth Factors/Hormones
    • PDGF
    • Cancer
    • Growth factors
    • PDGF
    • Cancer
    • Oncoproteins/suppressors
    • Oncoproteins
    • Growth factor receptors
    • Cardiovascular
    • Atherosclerosis
    • Vascular Inflammation
    • Inflammatory mediators

Target

  • Function

    Receptor that binds both PDGFA and PDGFB and has a tyrosine-protein kinase activity.
  • Tissue specificity

    Expressed in primary and metastatic colon tumors and in normal colon tissue. Tumors may express a different isoform to that found in normal tissue.
  • Involvement in disease

    Note=A chromosomal aberration involving PDGFRA is found in some cases of hypereosinophilic syndrome. Interstitial chromosomal deletion del(4)(q12q12) causes the fusion of FIP1L1 and PDGFRA (FIP1L1-PDGFRA).
  • Sequence similarities

    Belongs to the protein kinase superfamily. Tyr protein kinase family. CSF-1/PDGF receptor subfamily.
    Contains 5 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 1 protein kinase domain.
  • Cellular localization

    Membrane.
  • Target information above from: UniProt accession P16234 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt

Associated products

  • KO cell lines

    • Human PDGFRA knockout SHSY-5Y cell lysate (ab275522)
  • Related Products

    • Anti-PDGFR alpha antibody [EPR5480] (ab134123)
    • Anti-PDGFR alpha antibody [EPR22059-270] (ab203491)
    • Anti-PDGFR alpha antibody [EPR22059-270] - BSA and Azide free (ab234965)
    • Anti-PDGFR alpha antibody [EPR5480] - BSA and Azide free (ab248689)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab275335 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: 122 kDa.
ICC
Use at an assay dependent concentration.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 122 kDa.
ICC
Use at an assay dependent concentration.

Images

  • Western blot - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
    Western blot - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
    All lanes : Anti-PDGFR alpha antibody [EPR22059-270] (ab203491) at 1/1000 dilution

    Lane 1 : Wild-type SH-SY5Y cell lysate
    Lane 2 : PDGFRA knockout SH-SY5Y cell lysate

    Lysates/proteins at 40 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 122 kDa
    Observed band size: 150 kDa
    why is the actual band size different from the predicted?



    Lanes 1 - 2: Merged signal (red and green). Green - ab203491 observed at 150 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

    ab203491 was shown to react with PDGFR alpha in wild-type SH-SY5Y cells in Western blot with loss of signal observed in PDGFRA knockout cell line ab275335 (knockout cell lysate ab275522). Wild-type SH-SY5Y and PDGFRA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab203491 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  • Immunocytochemistry - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
    Immunocytochemistry - Human PDGFRA knockout SH-SY5Y cell line (ab275335)

    ab203491 staining PDGFR alpha in wild-type SH-SY5Y cells (top panel) and PDGFRA knockout SH-SY5Y cells (bottom panel) (ab275335). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab203491 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
    Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

  • Western blot - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
    Western blot - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
    All lanes : Anti-PDGFR alpha antibody [EPR5480] (ab134123) at 1/1000 dilution

    Lane 1 : Wild-type SH-SY5Y cell lysate
    Lane 2 : PDGFRA knockout SH-SY5Y cell lysate

    Lysates/proteins at 40 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 122 kDa
    Observed band size: 180 kDa why is the actual band size different from the predicted?



    Lanes 1 - 2: Merged signal (red and green). Green - ab134123 observed at 180 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

    ab134123 was shown to react with PDGFR alpha in wild-type SH-SY5Y cells in Western blot with loss of signal observed in PDGFRA knockout cell line ab275335 (knockout cell lysate ab275522). Wild-type SH-SY5Y and PDGFRA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab134123 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  • Sanger Sequencing - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
    Sanger Sequencing - Human PDGFRA knockout SH-SY5Y cell line (ab275335)
    Allele-1: 67 bp deletion in exon 3

Protocols

  • Hemocytometer protocol
  • Mammalian cell tissue culture techniques protocol

Click here to view the general protocols

Datasheets and documents

    • Datasheet
    • SDS
  • References (0)

    Publishing research using ab275335? Please let us know so that we can cite the reference in this datasheet.

    ab275335 has not yet been referenced specifically in any publications.

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