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).