Overview

  • Product name

    Anti-Occludin antibody [EPR20992]
    See all Occludin primary antibodies
  • Description

    Rabbit monoclonal [EPR20992] to Occludin
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, 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

    • WB: Human colon lysate; MDCK, PC-3, HEK-293 and Caco-2 whole cell lysates; Rat brain lysate; Mouse brain and uterus lysates. IHC-P: Human colon and breast tissues; Mouse and rat kidney tissues. ICC/IF: Caco-2 and MDCK cells. Flow Cyt: Caco-2 cells. IP: Caco-2 whole cell lysate.
  • General notes

    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

    Preservative: 0.01% Sodium azide
    Constituents: 0.05% BSA, 40% Glycerol, 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 ab216327 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. Detects a band of approximately 65, 53, 25, 23 kDa (predicted molecular weight: 59 kDa).
IHC-P 1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/100.
Flow Cyt 1/60.
IP 1/40.

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 at 40 µg
    Lane 2 : OCLN (Occludin) knockout HAP1 whole cell lysate at 40 µg
    Lane 3 : HeLa whole cell lysate (Low Occludin expression) at 20 µg
    Lane 4 : HepG2 whole cell lysate lysate (High Occludin expression) at 20 µg

    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.

  • 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.

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

  • Lane 1 : Anti-Occludin antibody [EPR8208] (ab167161) at 1/5000 dilution
    Lane 2 : Anti-Occludin antibody (ab31721) at 1/1000 dilution
    Lane 3 : Anti-Occludin antibody [EPR20992] (ab216327) at 1/5000 dilution

    All lanes : Recombinant Human Occludin protein (ab114189)

    Lysates/proteins at 0.025 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 59 kDa
    Observed band size: 85 kDa
    why is the actual band size different from the predicted?



    Blocking and diluting buffer: 5% NFDM/TBST

    Exposure time:
    5.5 seconds

  • 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.

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

    Lane 1 : Human colon lysate at 20 µg
    Lane 2 : MDCK (canine kidney cell line) cell lysate at 20 µg
    Lane 3 : PC-3 (human prostate adenocarcinoma cell line) cell lysate at 20 µg
    Lane 4 : HEK-293 (human epithelial cell line from embryonic kidney) cell lysate at 20 µg
    Lane 5 : Caco-2 (human colorectal adenocarcinoma cell line) cell lysate at 10 µg

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Predicted band size: 59 kDa
    Observed band size: 25,53,65 kDa why is the actual band size different from the predicted?



     

    Exposure time : Lanes 1-4: 3 minutes; lane 5: 1 minute.

    Blocking/Dilution buffer: 5% NFDM/TBST.

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

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

    Lane 1 : Rat brain lysate
    Lane 2 : Mouse brain lysate
    Lane 3 : Mouse uterus lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Predicted band size: 59 kDa
    Observed band size: 23,53,65 kDa why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

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

  • 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.

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

  • 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.

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

  • 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.

    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.

  • 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.

  • 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 1/10,000 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).

References

This product has been referenced in:

  • Hao FL  et al. The neurovascular protective effect of alogliptin in murine MCAO model and brain endothelial cells. Biomed Pharmacother 109:181-187 (2019). Read more (PubMed: 30396075) »
  • Shi J & Zhao XH Effect of caseinate glycation with oligochitosan and transglutaminase on the intestinal barrier function of the tryptic caseinate digest in IEC-6 cells. Food Funct 10:652-664 (2019). Read more (PubMed: 30652176) »
See all 12 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Testis, adult)
Permeabilization
Yes - 0.1% Triton X-100 in PBS
Specification
Testis, adult
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 20°C
Fixative
Paraformaldehyde

Mr. Bryan Niedenberger

Verified customer

Submitted Apr 28 2019

Application
Western blot
Sample
Mouse Tissue lysate - whole (Mouse brain)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-tris in MES)
Loading amount
10 µg
Specification
Mouse brain
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 15 2018

Application
Western blot
Sample
Mouse Tissue lysate - whole (Mouse brain)
Gel Running Conditions
Reduced Denaturing (4-12% Bis-tris in MOPS)
Loading amount
10 µg
Specification
Mouse brain
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 27 2017

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