Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Occludin antibody [EPR20992] - BSA and Azide free (ab224526)

Overview

  • Product name

    Anti-Occludin antibody [EPR20992] - BSA and Azide free
    See all Occludin primary antibodies
  • Description

    Rabbit monoclonal [EPR20992] to Occludin - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WB, Flow Cyt, ICC/IF, IPmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Dog, Human
  • Immunogen

    Recombinant fragment within Human Occludin aa 350 to the C-terminus. The exact sequence is proprietary.
    Database link: Q16625

  • Positive control

    • IHC-P: Human colon tissue.
  • General notes

    Ab224526 is the carrier-free version of ab216327. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    ab224526 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    Constituent: PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR20992
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab224526 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
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB Use at an assay dependent concentration. Detects a band of approximately 65, 53, 25, 23 kDa (predicted molecular weight: 59 kDa).
Flow Cyt Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
IP Use at an assay dependent concentration.

Target

  • Function

    May play a role in the formation and regulation of the tight junction (TJ) paracellular permeability barrier. It is able to induce adhesion when expressed in cells lacking tight junctions.
  • Tissue specificity

    Localized at tight junctions of both epithelial and endothelial cells. Highly expressed in kidney. Not detected in testis.
  • Involvement in disease

    Defects in OCLN are the cause of band-like calcification with simplified gyration and polymicrogyria (BLCPMG) [MIM:251290]; also known as pseudo-TORCH syndrome. BLCPMG is a neurologic disorder with characteristic clinical and neuroradiologic features that mimic intrauterine TORCH infection in the absence of evidence of infection. Affected individuals have congenital microcephaly, intracranial calcifications, and severe developmental delay.
  • Sequence similarities

    Belongs to the ELL/occludin family.
    Contains 1 MARVEL domain.
  • Domain

    The C-terminal is cytoplasmic and is important for interaction with ZO-1. Sufficient for the tight junction localization. Involved in the regulation of the permeability barrier function of the tight junction (By similarity). The first extracellular loop participates in an adhesive interaction.
  • Post-translational
    modifications

    Phosphorylated upon DNA damage, probably by ATM or ATR. Dephosphorylated by PTPRJ. The tyrosine phosphorylation on Tyr-398 and Tyr-402 reduces its ability to interact with TJP1.
  • Cellular localization

    Membrane. Cell junction > tight junction.
  • Information by UniProt
  • Database links

  • Alternative names

    • BLCPMG antibody
    • FLJ08163 antibody
    • FLJ18079 antibody
    • FLJ77961 antibody
    • FLJ94056 antibody
    • MGC34277 antibody
    • Occludin antibody
    • Ocln antibody
    • OCLN_HUMAN antibody
    • Phosphatase 1 regulatory subunit 115 antibody
    • PPP1R115 antibody
    • PTORCH1 antibody
    • Tight junction protein occludin antibody
    see all

Images

  • All lanes : Anti-Occludin antibody [EPR20992] (ab216327) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : OCLN (Occludin) knockout HAP1 whole cell lysate
    Lane 3 : HeLa whole cell lysate (Low Occludin expression)
    Lane 4 : HepG2 whole cell lysate lysate (High Occludin expression)

    Lysates/proteins at 40 µg per lane.

    Predicted band size: 59 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab216327 observed at 59 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab216327 was shown to recognize Occludin in wild-type HAP1 cells as signal was lost at the expected MW in OCLN (Occludin) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and OCLN (Occludin) knockout samples were subjected to SDS-PAGE. Ab216327 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at  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.

    Occludin expression in HeLa is expected to be negative.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

  • Immunohistochemical analysis of paraffin-embedded human breast tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on human breast is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MDCK (canine kidney cell line) cells labeling Occludin with ab216327 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membrane staining on MDCK (NBL-2) cell line.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Caco-2 (human colorectal adenocarcinoma cell line) cell line labeling Occludin with ab216327 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

  • Occludin was immunoprecipitated from 0.35 mg of Caco-2 (human colorectal adenocarcinoma cell line) whole cell lysate with ab216327 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab216327 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

    Lane 1: Caco-2 whole cell lysate 10 µg (Input). 

    Lane 2: ab216327 IP in Caco-2 whole cell lysate. 

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab216327 in Caco-2 whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 1 second.

    The molecular weight observed is consistent with what has been described in the literature (PMID: 18647175, PMID: 19821483).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Caco-2 (human colorectal adenocarcinoma cell line) cells labeling Occludin with ab216327 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membrane staining on Caco-2 cell line.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

  • Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on distal tubules of rat kidney is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on distal tubules of mouse kidney is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on human colon is observed (PMID: 24268521). Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216327).

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

References

ab224526 has not yet been referenced specifically in any publications.

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