- 701825 - Invitrogen Antibodies TJP3 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of MCF-7 (Lane1), Caco2 (Lane 2) and HT-29 (Lane 3) scraped on ice. The blots were probed with Anti-ZO-3 Recombinant Rabbit Monoclonal Antibody (Product # 701825, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 140 kDa band corresponding to ZO-3 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with wet transfer system (Product # EI9051). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence was performed on fixed and permeabilized Caco2 cells for detection of ZO-3 using Anti-ZO-3 Recombinant Rabbit Monoclonal Antibody (Product # 701825, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of ZO-3 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating membrane localization of ZO-3 Panel e) represents control cells with no primary Antibody to assess background.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Tight junction protein ZO-3 was performed using NIH:OVCAR-3 cells at 100% confluency. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with ZO-3 Recombinant Rabbit Monoclonal Antibody (24H3L15) (Product # 701825) at 1:200 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2500), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear, perinuclear, endosomal and junctional membrane localization. Panel d represents the merged image showing nuclear, perinuclear, endosomal and membrane junctional localization. Panel e represents low expression model SK-OV-3. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

701825