Recombinant Anti-Occludin antibody [EPR20992] (ab216327)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR20992] to Occludin
- Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cyt (Intra)
- Knockout validated
- Reacts with: Mouse, Rat, Dog, Human
Related conjugates and formulations
Overview
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Product name
Anti-Occludin antibody [EPR20992]
See all Occludin primary antibodies -
Description
Rabbit monoclonal [EPR20992] to Occludin -
Host species
Rabbit -
Specificity
This antibody is not suitable for ICC in mouse species
Not suitable for testing rat intestinal tissues
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Tested applications
Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cyt (Intra)more details -
Species reactivity
Reacts with: Mouse, Rat, Dog, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Human colon lysate; MDCK, PC-3, HEK-293 and Caco-2 whole cell lysates; Rat brain and lung lysate; Mouse colon, brain, lung and uterus lysates; bEnd.3 whole cell lysate. IHC-P: Human colon and breast tissues; human thyroid carcinoma; Mouse and rat kidney tissues. ICC/IF: Caco-2 and MDCK cells. Flow Cyt (intra): Caco-2 cells. IP: Caco-2 whole cell lysate.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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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
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 0.05% BSA, 40% Glycerol, PBS -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR20992 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
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 |
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WB | (3) |
1/1000. Detects a band of approximately 65, 53, 25, 23 kDa (predicted molecular weight: 59 kDa).
Please upload more of the lysate or use lower dilution of ab216327 when testing bEnd.3 and mouse intestinal tissues. ab216327 is not suitable for testing rat intestinal tissues. |
IHC-P | (3) |
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ICC/IF |
1/100.
This antibody is not suitable for ICC in mouse species. |
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IP |
1/40.
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Flow Cyt (Intra) |
1/60.
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Notes |
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WB
1/1000. Detects a band of approximately 65, 53, 25, 23 kDa (predicted molecular weight: 59 kDa). Please upload more of the lysate or use lower dilution of ab216327 when testing bEnd.3 and mouse intestinal tissues. ab216327 is not suitable for testing rat intestinal tissues. |
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. This antibody is not suitable for ICC in mouse species. |
IP
1/40. |
Flow Cyt (Intra)
1/60. |
Target
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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
modificationsPhosphorylated 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
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Database links
- Entrez Gene: 403844 Dog
- Entrez Gene: 100506658 Human
- Entrez Gene: 18260 Mouse
- Entrez Gene: 83497 Rat
- Omim: 602876 Human
- SwissProt: Q28269 Dog
- SwissProt: Q16625 Human
- SwissProt: Q61146 Mouse
see all -
Alternative names
- BLCPMG antibody
- FLJ08163 antibody
- FLJ18079 antibody
see all
Images
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
Tissue Microarrays stained for Anti-Occludin antibody [EPR20992] using ab216327 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The sections were incubated with ab216327 at 4°C overnight used at 1:2000 dilution (1.05 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB secondary antibody (ab209101). Counterstain was Hematoxylin.
Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).
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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 kDaLanes 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. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma labeling Occludin with ab216327 at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880), ready to use. Counter stained with Hematoxylin. Antigen retrieval was heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).
Secondary antibody only control: ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).
Positive staining on human thyroid carcinoma. The section was incubated with ab216327 at 4°C overnight.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
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: ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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All lanes : Anti-Occludin antibody [EPR20992] (ab216327) at 1/1000 dilution
Lane 1 : Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : bEnd.3 (Mouse brain endothelioma) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
Predicted band size: 59 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?
Exposure time: 60 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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All lanes : Anti-Occludin antibody [EPR20992] (ab216327) at 1/1000 dilution
Lane 1 : Mouse colon tissue lysate
Lane 2 : Mouse brain tissue lysate
Lane 3 : Mouse lung tissue lysate
Lane 4 : Rat colon tissue lysate
Lane 5 : Rat brain tissue lysate
Lane 6 : Rat lung tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
Predicted band size: 59 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?
Exposure time: 100 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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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.
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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 -
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).
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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 minutesBlocking/Dilution buffer: 5% NFDM/TBST.
The molecular weight observed is consistent with what has been described in the literature (PMID: 18647175, PMID: 19821483).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (ab216327)
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: ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (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: ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] (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: ab209101 (Rabbit specific IHC polymer detection kit HRP/DAB).
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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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.
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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).
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Intracellular 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.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (165)
ab216327 has been referenced in 165 publications.
- Huang YM et al. Glucagon-Like Peptide-2 Ameliorates Age-Associated Bone Loss and Gut Barrier Dysfunction in Senescence-Accelerated Mouse Prone 6 Mice. Gerontology 69:428-449 (2023). PubMed: 36470214
- Hill AA et al. Acute high-fat diet impairs macrophage-supported intestinal damage resolution. JCI Insight 8:N/A (2023). PubMed: 36538527
- Xu P et al. Intestinal microbiota analysis and network pharmacology reveal the mechanism by which Lianhua Qingwen capsule improves the immune function of mice infected with influenza A virus. Front Microbiol 13:1035941 (2022). PubMed: 36504796
- Harati R et al. miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability. PLoS One 17:e0262152 (2022). PubMed: 35025943
- Liu N et al. Lactobacillus rhamnosus Ameliorates Multi-Drug-Resistant Bacillus cereus-Induced Cell Damage through Inhibition of NLRP3 Inflammasomes and Apoptosis in Bovine Endometritis. Microorganisms 10:N/A (2022). PubMed: 35056585