Overview

  • Product name

    Anti-PODXL antibody [EPR9518]
    See all PODXL primary antibodies
  • Description

    Rabbit monoclonal [EPR9518] to PODXL
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cytmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant fragment corresponding to Human PODXL aa 300-500.
    Database link: O00592

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    O00592

  • Positive control

    • Raji, HeLa, and Human fetal kidney lysates, Human kidney tissue
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab150358 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 1/1000 - 1/10000. Detects a band of approximately 165 kDa (predicted molecular weight: 58 kDa).
IHC-P 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

For unpurified use at 1/250 - 1/500.

ICC/IF 1/100.

For unpurified use at 1/500.

Flow Cyt 1/250.
  • Application notes
    Is unsuitable for IP.
  • 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).
    • Information by UniProt
    • Database links

    • Form

      There are 2 isoforms produced by alternative splicing.
    • Alternative names

      • GCTM-2 antigen antibody
      • Gp2 antibody
      • Gp200 antibody
      • MGC138240 antibody
      • PC antibody
      • PCLP antibody
      • PCLP-1 antibody
      • PCLP1 antibody
      • Pcx antibody
      • Podocalyxin antibody
      • Podocalyxin like antibody
      • Podocalyxin like protein antibody
      • Podocalyxin-like protein 1 antibody
      • Podxl antibody
      • PODXL_HUMAN antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling PODXL with purified ab150358 at 1:1000 dilution (0.44 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

    • Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
      Lane 2: PODXL knockout HAP1 whole cell lysate (20 µg)
      Lane 3: Raji whole cell lysate (20 µg)
      Lane 4: HeLa whole cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab150358 observed at 160 kDa. Red - loading control, ab9484, observed at 37 kDa.

      ab150358 was shown to specifically react with PODXL in wild-type cells as signal was lost in PODXL knockout cells. Wild-type and PODXL knockout samples were subjected to SDS-PAGE. Ab150358 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

    • Anti-PODXL antibody [EPR9518] (ab150358) at 1/10000 dilution (purified) + Human fetal kidney lysates at 15 µg

      Secondary
      Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

      Predicted band size: 58 kDa
      Observed band size: 165 kDa
      why is the actual band size different from the predicted?



      Blocking and diluting buffer: 5% NFDM/TBST

    • Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling PODXL with purified ab150358 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue). Secondary Only Control: PBS was used instead of the primary antibody as the negative control. 

    • All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution (unpurified)

      Lane 1 : Raji lysate
      Lane 2 : HeLa lysate
      Lane 3 : Human fetal kidney lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 58 kDa
      Observed band size: 165 kDa why is the actual band size different from the predicted?

    • Immunohistochemical analysis of paraffin embedded Human kidney tissue labelling PODXL with unpurified ab150358 antibody at a dilution of 1/100. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

    • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling PODXL with purified ab150358 at 1/250 dilution (red). The secondary antibody was Goat anti rabbit IgG (Alexa Fluor® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

    • Unpurified ab150358 staining PODXL in human kidney tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue samples were fixed with formaldehyde, cut into 20 micron slices, permeablized with 0.05% tween-20 and blocked for 60 minutes at 25°C. Antigen retrieval was by heat mediation. The sample was incubated with primary antibody at a dilution of 1/250 at 25°C for 1 hour. An Alexa Fluor® 488-conjugated donkey anti-rabbit polyclonal (1/1000) was used as the secondary antibody, at a dilution of 1/1200.

      See Abreview

    • Immunohistochemical analysis of paraffin embedded Human Hepatocellular carcinoma vessels tissue using unpurified ab150358 showing +ve staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

    • Immunohistochemical analysis of paraffin embedded Human Breast adenocarcinoma tissue using unpurified ab150358 showing +ve staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

    • Immunohistochemical analysis of paraffin embedded Human Endometrial carcinoma tissue using unpurified ab150358 showing +ve staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

    • Immunohistochemical analysis of paraffin embedded Human Skeletal muscle tissue using unpurified ab150358 showing -ve staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

    • Immunohistochemical analysis of paraffin embedded Normal Human Kidney tissue using unpurified ab150358 showing +ve staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

    • Immunohistochemical analysis of paraffin embedded Human Glioma tissue using unpurified ab150358 showing +ve staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

    References

    This product has been referenced in:

    • Zhi Q  et al. Podocalyxin-like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein. Cancer Sci 110:118-134 (2019). Read more (PubMed: 30407695) »
    • Zhang LH  et al. Early podocyte injury and elevated levels of urinary podocyte-derived extracellular vesicles in swine with metabolic syndrome: role of podocyte mitochondria. Am J Physiol Renal Physiol 317:F12-F22 (2019). Read more (PubMed: 31042059) »
    See all 3 Publications for this product

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Kidney)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Antigen Retrieval Buffer (100X Citrate Buffer pH 6.0) (ab94674)
    Permeabilization
    Yes - 0.05% tween 20
    Specification
    Kidney
    Blocking step
    Sea Block as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 22°C
    Fixative
    10%NBF

    Mr. David Ivancic

    Verified customer

    Submitted May 24 2018

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (adult kidney)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: commercial - ZYTOMED's OmniPrep (pH 9)
    Permeabilization
    Yes - 0.05% tween-20
    Specification
    adult kidney
    Blocking step
    commercial - Invitrogen's CAS-Block as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
    Fixative
    Formaldehyde

    Abcam user community

    Verified customer

    Submitted Dec 11 2015

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