Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [2]
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Validation data
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- Product number
- 720298 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- ZO-3 Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant full-length protein
- Description
- These Polyclonal antibodies are of rabbit origin developed by immunizing animals with proteins or peptides. The polyclonal antibody is purified by affinity purification from the rabbit sera generated after immunizing the rabbits with a specific type of protein or peptide. The purified antibody is tested for its functionality in various relevant research applications. The antibody is developed for Research Use Only and is non-hazardous or non-infectious in nature.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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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 CaCo-2 (Lane 1), MCF7 (Lane 2), HT-29 (Lane 3) and HCT-116 (Lane 4). The blots were probed with Anti-ZO-3 Rabbit Polyclonal Antibody (Product # 720298, 0.5-1 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 iBlot® Dry Blotting System (Product # IB21001). 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 Rabbit Polyclonal Antibody (Product # 720298, 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.
- 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 Polyclonal Antibody (Product # 720298) 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 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.