Antibodies, Proteins, Kits and Reagents for Life Science

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

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 - BSA and Azide free
Suitable for: IHC-P, WB, Flow Cyt, ICC/IF, IP
Knockout validated
Reacts with: Mouse, Rat, Dog, Human

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 & Species

Application	Species
Flow Cyt	Human
ICC/IF	Dog
IHC-P	Human
IP	Human
WB	Human

See all applications and species data

Immunogen

Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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
ab224526 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

Maxpar® is a trademark of Fluidigm Canada Inc.
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
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C. Do Not Freeze.
Storage buffer

pH: 7.2
Constituent: PBS
Carrier free

Yes
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR20992
Isotype

IgG
Research areas

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

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).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (ab224526)

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.
Immunocytochemistry/ Immunofluorescence - Anti-Occludin antibody [EPR20992] - BSA and Azide free (ab224526)

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

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

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).
Immunocytochemistry/ Immunofluorescence - Anti-Occludin antibody [EPR20992] - BSA and Azide free (ab224526)

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).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (ab224526)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (ab224526)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Occludin antibody [EPR20992] - BSA and Azide free (ab224526)

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

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